CN114641504A - anti-B7H 4 antibody and double antibody and application thereof - Google Patents

anti-B7H 4 antibody and double antibody and application thereof Download PDF

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CN114641504A
CN114641504A CN202180006237.9A CN202180006237A CN114641504A CN 114641504 A CN114641504 A CN 114641504A CN 202180006237 A CN202180006237 A CN 202180006237A CN 114641504 A CN114641504 A CN 114641504A
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amino acid
acid sequence
variable region
chain variable
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CN114641504B (en
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吴晓东
王永强
陈飞
何进秋
贾鸽子
赵楚楚
陈庆芳
戎一平
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Nuona Biological Suzhou Co ltd
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Harbour Biomed Shanghai Co Ltd
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Abstract

Provided are a B7H 4-targeting antibody or variant thereof, and a bispecific antibody comprising a B7H 4-targeting antibody, wherein the B7H 4-targeting antibody comprises a light chain variable region and a heavy chain variable region, and the variant has an amino acid mutation in the light chain variable region or the heavy chain variable region of the antibody and is capable of maintaining the function of the antibody. Antibodies targeting B7H4 have activity in binding to human B7H4 and cynomolgus monkey B7H4 without cross-reactivity with other B7 family members; in vivo and in vitro experiments show that the compound has good antitumor activity, T cell activation activity and internalization activity, and is more suitable for being used as ADC medicines. Also provides a bispecific antibody targeting B7H4 and CD3, which has longer half-life, retains good stability and hydrophilicity, and reduces toxicity on the premise of ensuring drug effect.

Description

anti-B7H4 antibody and double antibody and application thereof
This application claims priority from chinese patent application 202010618159.5 filed as 2020/6/30. The present application refers to the above-mentioned chinese patent application in its entirety.
Technical Field
The invention relates to the field of biological medicines, in particular to an anti-B7H4 antibody and a double antibody and application thereof.
Background
Breast cancer, ovarian cancer and endometrioma are common malignant tumors of women, wherein the incidence of the breast cancer is the first of the incidence of the female cancer, and the data of international agency for research on cancer IARC in 2018 show that the incidence of the breast cancer in the female cancer worldwide is 24.2%. For the treatment of these cancers, immunotherapy and targeted therapy are currently hot spots. For example, herceptin targeting Her2 has good therapeutic effects on Her2 positive breast cancer. For Triple Negative Breast Cancer (TNBC), few therapeutic approaches exist at present due to the lack of corresponding targets, mainly chemotherapy. Us FDA approved the PD-L1 inhibitor Atezolizumab in combination with albumin-bound paclitaxel for the treatment of metastatic Triple Negative Breast Cancer (TNBC) in month 3 2019, but PD-L1 is not highly expressed on triple negative breast cancer.
Immune checkpoint inhibitors are the most studied form of immunotherapy in breast cancer. Immune checkpoint molecules are often highly expressed in the tumor microenvironment, so that tumors can evade attack by the immune system by inhibiting T cell activation and inducing T cell depletion. The B7 family and the TNF family are two main co-stimulatory molecule families, and the B7 family has 10 molecules at present, namely CD80(B7.1), CD86(B7.2), B7H1(PD-L1/CD274), B7-DC (PD-L2/CD273), B7H2(ICOSL), B7H3(CD276), B7H4(B7S1/B7x/Vtcn1), B7H5(VISTA), B7H6 and B7H7(HHLA 2). Multiple members of the B7 family and their receptors have been shown to be immune checkpoints, such as PD-L1/PD1, CTLA4, and VISTA, among others.
B7H4 is a relatively new B7 family member, and although widely expressed in body cells at mRNA level, the protein level is very limited, and only in part of ductal epithelial cells of the body, such as mammary duct and lobule, oviduct epithelium, endometrium and other tissues, the expression is low. In contrast, B7H4 is abundantly expressed in a variety of tumor tissues, such as on tumor cells of breast cancer, particularly triple negative breast cancer, ovarian cancer, and endometrioma. From the point of expression profile, B7H4 can be considered as a highly specific tumor-associated antigen. On the other hand, B7H4 is a novel immune checkpoint molecule, and in vitro experiments demonstrate that B7H4 inhibits T cell proliferation, activation and cytokine production by interacting with its unknown T cell surface receptor. The tumor cells inhibit activation of T cells through high expression of B7H4 molecules and inhibitory macrophages of high expression of B7H4 molecules in a tumor microenvironment, so that immune evasion is realized. The expression profile of B7H4 on the tumor did not overlap with that of PD-L1. The treatment by antibody targeting of B7H4 and blocking of the negative regulation effect of B7H4 to reactivate the immune system are a promising means for treating B7H4 positive tumors.
Monoclonal antibodies against B7-H4, or drug conjugates, or bispecific antibodies are currently being developed by several pharmaceutical companies. Genentech, BMS, Jounce, junce, junsu haonex et al are in preclinical development, and the most rapidly progressing antibody against B7H4 by filprime is currently in phase I of the clinic, with its mechanism mainly activating T cells via ADCC and blocking immune checkpoints. However, a human anti-B7H4 antibody with high affinity, high selectivity and high biological activity is not available clinically.
Disclosure of Invention
In order to solve the technical problem that a human anti-B7H4 antibody with high affinity, high selectivity and high biological activity is lacked in the field, the invention provides a fully human anti-B7H4 antibody with high affinity, high selectivity and high biological activity by utilizing a specific and platinum human mouse platform, and a bispecific antibody of anti-B7H4 xCD 3 constructed based on the antibody, so as to be applied to the treatment of B7H4 positive tumors.
One of the technical schemes of the invention is as follows: providing an antibody or variant thereof targeting B7H4, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein:
the light chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 47. LCDR1, LCDR2 and LCDR3 shown at 54 and 64; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 21 and 35; or the like, or a combination thereof,
the light chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 48. LCDR1, LCDR2 and LCDR3 shown at 54 and 65; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 11. HCDR1, HCDR2 and HCDR3 shown at 22 and 36;
the variants have amino acid mutations in the light chain variable region and/or the heavy chain variable region of the antibody and maintain the function of the antibody. In the present invention, the amino acid mutation may be a deletion, substitution or addition of one or more amino acid residues in the original amino acid sequence, for example, CDR. Preferably, the amino acid mutation is an amino acid substitution, and the number of the amino acid substitutions is 1-3. And the mutated amino acid sequence has at least 85% sequence identity to the original amino acid sequence and retains or improves binding of the antibody to the antigen of interest; said at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95%, 96%, 97% or 98% sequence identity; most preferably at least 99% sequence identity. The antibody targeting B7H4 is also referred to herein as an anti-B7H4 antibody.
In specific embodiments, the antibody or variant thereof as described above, wherein said variant comprises an amino acid substitution at position 3 or4 of HCDR2, position 3 of HCDR3, and/or an amino acid substitution at position 4 of LCDR3 in the heavy chain variable region of said antibody.
Preferably, D at position 3 of the HCDR2 is replaced by G or E, and/or G at position 4 is replaced by a, and/or position 3 of the HCDR3 is replaced by G to a; the 4 th position of the LCDR3 is replaced by S, R or Q from N. That is, the light chain variable region comprises an amino acid sequence consisting of SEQ ID NOs: 48. LCDR1, LCDR2 and LCDR3 shown at 55 and 66; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 11. HCDR1, HCDR2 and HCDR3 shown at 22 and 36; or, the light chain variable region comprises an amino acid sequence consisting of SEQ ID NOs: 47. LCDR1, LCDR2 and LCDR3 shown at 54 and 67; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 23 and 35; or, the light chain variable region comprises an amino acid sequence consisting of SEQ ID NOs: 47. LCDR1, LCDR2 and LCDR3 shown at 54 and 67; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 24 and 35; or, the light chain variable region comprises an amino acid sequence consisting of SEQ ID NOs: 47. LCDR1, LCDR2 and LCDR3 shown at 54 and 69; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 9. 24 and 38, HCDR1, HCDR2, and HCDR 3.
In specific embodiments, the antibody or variant thereof, wherein the heavy chain variable region further comprises a heavy chain variable region framework region HFWR, and/or the light chain variable region further comprises a light chain variable region framework region LFWR, wherein the HFWR is the heavy chain variable region framework region of a human antibody and the LFWR is the light chain variable region framework region of a human antibody.
Preferably, the first and second liquid crystal films are made of a polymer,
the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 87; the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 77; or the like, or, alternatively,
the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 88; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79; or the like, or a combination thereof,
the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 89; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79; or the like, or, alternatively,
the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 90; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 80; or
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 90; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 81; or
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 93; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 83; or
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 91; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79, or a pharmaceutically acceptable salt thereof.
In the present invention, the amino acid mutation may also be deletion, substitution or addition of one or more amino acid residues in the original amino acid sequence such as FWR. Preferably, the amino acid mutation is an amino acid substitution, and the number of the amino acid substitutions is 1-3. And the mutated amino acid sequence has at least 85% sequence identity to the original amino acid sequence and retains or improves binding of the antibody to the antigen of interest; said at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95%, 96%, 97% or 98% sequence identity; most preferably at least 99% sequence identity.
In specific embodiments, the antibody or variant thereof described above, wherein the antibody further comprises a heavy chain constant region and/or a light chain constant region. Preferably, the heavy chain constant region of the antibody is selected from the group consisting of hIgG1, hIgG2, hIgG3 or hIgG4 and the light chain constant region is selected from the group consisting of a kappa chain or a lambda chain; more preferably, the variant has an amino acid substitution at position 239 and/or 332 of the Fc of the antibody, preferably the amino acid substitution is S239D and/or I332E.
In specific embodiments, the antibody or variant thereof, as described above, wherein the antibody is a full length antibody, Fab ', F (ab')2Fv, scFv, or a monoclonal antibody or a polyclonal antibody produced from the above antibody.
In specific embodiments, the antibody as described above, wherein said antibody comprises (1) a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 95; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 109; or, the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 98; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 112; or the like, or, alternatively,
the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 98; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 113; or the like, or, alternatively,
the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 99; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or, alternatively,
the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 100; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or a combination thereof,
the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 101; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 109; or the like, or, alternatively,
the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 102; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or, alternatively,
the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 106; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 117; or the like, or, alternatively,
the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 98; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 115; or the like, or, alternatively,
the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 103; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 112; or the like, or a combination thereof,
the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 103; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 115; or the like, or, alternatively,
(2) it comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 132. .
The term "antibody" may include immunoglobulins, which are tetrapeptide chain structures made up of two identical heavy chains and two identical light chains joined by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. In the five classes of igs, the second class of igs can have either kappa chains or lambda chains.
In the present application, the antibody light chain variable region described herein may further comprise a light chain constant region comprising a human kappa, lambda chain or variant thereof. In the present application, the antibody heavy chain variable region described herein may further comprise a heavy chain constant region comprising human IgG1, 2, 3, 4 or variants thereof.
Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 framework regions (FWRs) that are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and the heavy chain variable region (VH) is composed of 3 CDR regions and 4 FWR regions, and the sequence from the amino terminus to the carboxyl terminus is: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR 4.
In order to solve the above technical problems, the second technical solution of the present invention is: providing a bispecific antibody targeting B7H4, wherein it comprises a protein a functional region and a protein B functional region, wherein the protein a functional region is an antibody targeting B7H4 as described above; the B protein functional region is an antibody which is not targeted to B7H 4; preferably, the non-B7H 4-targeting antibody is a CD 3-targeting antibody; more preferably, the CD 3-targeting antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises amino acid sequences represented by SEQ ID NOs: 46. LCDR1, LCDR2 and LCDR3 shown at 53 and 63; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; or, the light chain variable region comprises an amino acid sequence consisting of SEQ ID NOs: 46. LCDR1, LCDR2 and LCDR3 shown at 53 and 63; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 10. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; even more preferably, in the CD 3-targeting antibody, the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 86; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; or, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 86; the VH of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78; or, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 86; the VH of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 84.
In specific embodiments, the bispecific antibody as described above, wherein the B protein functional region comprises a light chain variable region and a heavy chain variable region and the a protein functional region comprises a light chain variable region and a heavy chain variable region; wherein the content of the first and second substances,
in the B protein functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 64, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 21 and 35; or the like, or, alternatively,
in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 67, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 23 and 35; or the like, or, alternatively,
in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 67, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 24 and 35; or the like, or, alternatively,
in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 48. 54 and 65, LCDR1, LCDR2, and LCDR3, the heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 11. HCDR1, HCDR2 and HCDR3 shown at 22 and 36; or the like, or, alternatively,
in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 64, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 21 and 35; or the like, or, alternatively,
in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10. 20 and 34, HCDR1, HCDR2 and HCDR 3. In the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 67, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 23 and 35; or the like, or, alternatively,
in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 67, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 24 and 35; or the like, or, alternatively,
in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, a light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 48. 54 and 65, LCDR1, LCDR2, and LCDR3, the heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 11. 22 and 36, HCDR1, HCDR2, and HCDR 3.
In the bispecific antibody of the present invention, the protein B functional region comprises a light chain variable region and a heavy chain variable region, and the protein a functional region comprises a light chain variable region and a heavy chain variable region; wherein the content of the first and second substances,
in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 87 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 77; or the like, or, alternatively,
in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 80; or the like, or, alternatively,
in the B protein functional region, a light chain variable region comprises a light chain variable region shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 81; or the like, or, alternatively,
in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79; or the like, or, alternatively,
in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78, or a pharmaceutically acceptable salt thereof. In the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 87 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 77; or the like, or, alternatively,
in the B protein functional region, a light chain variable region comprises a light chain variable region shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 80; or the like, or, alternatively,
in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 81; or the like, or, alternatively,
in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79; or the like, or a combination thereof,
in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 84; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79, or a pharmaceutically acceptable salt thereof.
In specific embodiments, the bispecific antibody as described above, wherein the bispecific antibody is selected from the group consisting of:
(1) polypeptide chain-1 of structure n '- [ VL ] -CL-c', polypeptide chain-2 of structure n '- [ VH ] -CH1-h-CH2-CH 3-c', and polypeptide chain-3 of n '- { VH-L-VL } -h-CH2-CH 3-c' or n '- { VL-L-VH } -h-CH2-CH 3-c'; wherein, the polypeptide chain-1 and the polypeptide chain-2 are protein A functional regions, and the polypeptide chain-3 is a protein B functional region, or, the polypeptide chain-1 and the polypeptide chain-2 are protein B functional regions, and the polypeptide chain-3 is a protein A functional region;
(2) polypeptide chain-1 of structure n '- [ VL ] -CL-c', polypeptide chain-2 of n '- [ VH ] -CH1-h-CH2-CH 3-c', polypeptide chain-3 of n '- { VH } -CH1-h-CH2-CH 3-c' and polypeptide chain-4 of n '- { VL } -CL-c', or polypeptide chain-1 of structure n '- [ VH ] -CH-c', polypeptide chain-2 of structure n '- [ VL ] -CL-h-CH2-CH 3-c', polypeptide chain-3 of structure n '- { VH } -CH1-h-CH2-CH 3-c' and polypeptide chain-4 of structure n '- { VL } -CL-c'; wherein, the polypeptide chain-1 and the polypeptide chain-2 are protein A functional regions, and the polypeptide chain-3 and the polypeptide chain-4 are protein B functional regions, or, the polypeptide chain-1 and the polypeptide chain-2 are protein B functional regions, and the polypeptide chain-3 and the polypeptide chain-4 are protein A functional regions;
(3) polypeptide chain-1 of structure n '- [ VH ] -CH 1-c', n '- { VH } -CH1-L- [ VL ] -CL-h-CH2-CH 3-c' or n '- { VH } -CH1-h-CH2-CH3-L- [ VL ] -CL-c' -polypeptide chain-2, n '- { VH } -CH1-h-CH2-CH 3-c' -polypeptide chain-3 and n '- { VL } -CL-c' -polypeptide chain-4; wherein, the polypeptide chain-1 is a functional region A, the polypeptide chain-2 comprises a functional region A protein and a functional region B protein from n 'to c' or comprises a functional region B protein and a functional region A protein from n 'to c', and the polypeptide chain-3 and the polypeptide chain-4 are functional regions B; or, the polypeptide chain-1 is a B functional region, the polypeptide chain-2 sequentially comprises a B protein functional region and an A protein functional region from n 'to c' or sequentially comprises a A protein functional region and a B protein functional region from n 'to c', and the polypeptide chain-3 and the polypeptide chain-4 are A functional regions;
(4) polypeptide chain-1 of structure n '- [ VH ] -CH1-L1- { VL-L2-VH } -h-CH2-CH 3-c' or n '- [ VH ] -CH1-L1- { VH-L2-VL } -h-CH2-CH 3-c', polypeptide chain-2 of n '- [ VH ] -CH1-h-CH2-CH 3-c' and polypeptide chain-3 of n '- [ VL ] -CL-c'; wherein said polypeptide chain-1 comprises a protein A functional region and a protein B functional region, and said polypeptide chains-2 and 3 are protein B functional regions, or said polypeptide chain-1 comprises a protein A functional region and a protein B functional region, and said polypeptide chains-2 and 3 are protein A functional regions; preferably, the polypeptide chain-1 is a functional B region and a functional A region in sequence from n 'to c', or a functional A region and a functional B region in sequence from n 'to c';
(5) polypeptide chain-1 of n '- [ VH ] -CH1-h-CH2-CH 3-c' and polypeptide chain-2 of n '- [ VL ] -CL-c' having the structure n '- [ VH ] -CH2-CH3-L1- { VL-L2-VH } -c' or n '- [ VH ] -CH1-h-CH2-CH3-L1- { VH-L2-VL } -c'; wherein, the polypeptide chain-1 comprises a protein A functional region and a protein B functional region, and the polypeptide chains-2 and 3 are protein B functional regions, or, the polypeptide chain-1 comprises a protein A functional region and a protein B functional region, and the polypeptide chains-2 and 3 are protein A functional regions.
Where N 'represents the amino terminus (also designated N-terminus), C' represents the carboxy terminus (also designated C-terminus) of the polypeptide chain, h represents the hinge region, L, L1 or L2 represents a linker (or linker), and a suitable prior art linker (L) is represented by the repeated G4S amino acid sequence or a variant thereof. For example, a polypeptide having an amino acid sequence (G4S)4Or (G)4S) 3Linkers, but variants thereof may also be used, e.g. the G4Where one of the G' S of S is replaced by Q, e.g. the second or third G is replaced by Q, preferred linker sequences of the invention are as set out in SEQ ID NO: 133-135. "-" represents a polypeptide bond linking different structural regions or serves to separate different structural regions.
In the present invention, "[ ]", "{ }" respectively represent different functional regions or structures; for example, { VL-L-VH }, { VH-L-VL } represents the scFv structure, and [ VH ]/{ VH }, [ VL ]/{ VL } represents the heavy chain variable region and the light chain variable region of the Fab structure, respectively. When VH is A, B protein domain, VH _ A (i.e. heavy chain variable region is A protein domain) or VH _ B (i.e. heavy chain variable region is B protein domain); similarly, when VL is A, B protein domains, VL can be expressed as VL _ A (i.e., light chain variable region is protein A domain) or VL _ B (i.e., light chain variable region is protein B domain). In FIG. 28, "[ ]" { } "is removed for convenience of mapping, so VL _ B-L-VH _ A (light chain variable region is the B protein functional region, heavy chain variable region is the A protein functional region, L is a linker connecting VL _ B and VH _ A), VH _ B-L-VL _ A (heavy chain variable region is the B protein functional region, light chain variable region is the A protein functional region, L is a linker connecting VH _ B and VL _ A, see, e.g., polypeptide chain-3 of structure (1)) equally represent scFv structures, while VL _ B or VH _ A alone (see, e.g., polypeptide chain-1 or polypeptide chain-2 of structure (1)) still represent the light chain variable region of the B protein functional region and the heavy chain variable region of the A protein functional region, respectively, in Fab structures. In the present invention, polypeptide chains-1, -2, -3 or-4 represent only the kind of polypeptide chains, and the number of each polypeptide chain can be 1 or 2 when the bispecific antibody is actually constituted by polypeptide chains, for example, in structures (5) and (6), the number of polypeptide chains-1, -2, -3 is 1, and the number of polypeptide chains-4 is 2; in structures (7), (8), (9) and (10), the number of polypeptide chains-1, -2 is 1 and the number of polypeptide chains-3 is 2. In the present invention, polypeptide chain-1 is also referred to as a first polypeptide chain, polypeptide chain-2 is also referred to as a second polypeptide chain, polypeptide chain-3 is also referred to as a third polypeptide chain, and polypeptide chain-4 is also referred to as a fourth polypeptide chain.
In some preferred embodiments, the bispecific antibody is selected from the group consisting of:
(1) the bispecific antibody comprises three polypeptide chains: wherein the first polypeptide chain comprises a sequence as set forth in SEQ ID NO: 109; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 114; the second polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 120; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 114; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 115; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 122; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 126, or a pharmaceutically acceptable salt thereof; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 127; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109, or a pharmaceutically acceptable salt thereof; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 110; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 128; the third polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 129; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 110; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 130; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 129;
(2) the bispecific antibody comprises four polypeptide chains: wherein the first polypeptide chain comprises a sequence as set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 118; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 109; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 120; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 121; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 122; the fourth polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 115; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 124; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 109; or the like, or, alternatively,
the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 109, or a pharmaceutically acceptable salt thereof.
The functional regions of the proteins of the invention may in some cases be Fab, scFv or VH, and in other cases may also be f (ab)2 or full length antibodies, also referred to in some cases as antibodies or antigen binding proteins or binding proteins. In the present invention, the "Fab structure" or "Fab fragment" consists of one light chain and one heavy chain of CH1 and the variable region. The heavy chain of a Fab fragment is unable to form a disulfide bond with another Fab heavy chain molecule. The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody; the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains. A "Fab ' fragment" then contains a light chain and a portion of a heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab ' fragments to form the F (ab ') 2 fragment. The "F (ab') 2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus a F (ab ') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The term "Fv" means an antibody fragment consisting of the VL and VH domains toward a single arm of an antibody, but lacking the constant region.
In the present invention, the scFv (single chain antibody) can be a single chain antibody conventional in the art, and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids. Wherein the VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain. Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH, or n '-VL-L-VH-c' or n '-VH-L-VL-c'.
In order to solve the technical problems, the third technical scheme of the invention is as follows: there is provided a chimeric antigen receptor comprising an antibody according to one embodiment of the present invention or a bispecific antibody according to a second embodiment of the present invention.
In the present application, the antibodies or bispecific antibodies can be used to prepare Chimeric Antigen Receptors (CARs) and the like to modify them on cells such as T cells or NK cells. The chimeric antigen receptor may be one that is conventional in the art, including, for example, one that utilizes the above-described antibody in the form of an scFv as an extracellular antigen-binding domain.
Therefore, to solve the above technical problems, the fourth technical solution of the present invention is: providing a genetically modified cell comprising an antibody according to one of the embodiments of the present invention; the cell is preferably a eukaryotic cell, more preferably an isolated human cell, even more preferably an immune cell such as a T cell (e.g. in the form of CAR-T), or an NK cell such as the NK92 cell line.
In order to solve the above technical problems, the fifth technical solution of the present invention is: there is provided an isolated nucleic acid encoding an antibody as described above or a bispecific antibody as described above or a chimeric antigen receptor as described in the third aspect of the invention.
The preparation method of the nucleic acid is a preparation method which is conventional in the field, and preferably comprises the following steps: obtaining the nucleic acid molecule coding the antibody by a gene cloning technology, or obtaining the nucleic acid molecule coding the antibody by an artificial complete sequence synthesis method.
Those skilled in the art know that the base sequence encoding the amino acid sequence of the above antibody may be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homolog. The polynucleotide homologue of the present invention may be produced by substituting, deleting or adding one or more bases of a gene encoding the antibody sequence within a range in which the activity of the antibody is maintained.
In order to solve the technical problems, the sixth technical scheme of the invention is as follows: an expression vector is provided comprising an isolated nucleic acid as described above.
The recombinant expression vector can be obtained by methods conventional in the art, namely: the nucleic acid molecules described herein are constructed by ligating them to various expression vectors. The expression vector is any vector conventionally used in the art so long as it can carry the aforementioned nucleic acid molecule.
Preferably, the expression vector comprises a eukaryotic cell expression vector and/or a prokaryotic cell expression vector.
In order to solve the above technical problems, the seventh technical solution of the present invention is: a transformant is provided comprising the isolated nucleic acid or expression vector as described above.
The preparation method of the transformant may be a preparation method conventional in the art, for example: the recombinant expression vector is transformed into a host cell to obtain the recombinant expression vector. The host cell of the transformant is a variety of host cells which are conventional in the art, as long as the recombinant expression vector is stably self-replicating and the nucleic acid carried by the recombinant expression vector can be efficiently expressed. Preferably, the host cell is a prokaryotic cell, preferably an e.coli cell such as TG1, BL21 (expressing a single chain antibody or Fab antibody), and/or a eukaryotic cell, preferably an HEK293 cell or a CHO cell (expressing a full length IgG antibody). The recombinant expression plasmid is transformed into a host cell to obtain a recombinant expression transformant of the present invention. Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.
To solve the above technical problems, an eighth technical solution of the present invention is: provided is a method for producing an antibody or bispecific antibody targeting B7H4, wherein the method for producing comprises the steps of: culturing the transformant according to the seventh embodiment of the present invention, and obtaining the antibody or bispecific antibody targeting B7H4 from the culture.
To solve the above technical problems, the ninth technical solution of the present invention is: there is provided an antibody drug conjugate comprising an antibody moiety comprising an antibody according to one embodiment of the present invention, a bispecific antibody according to the second embodiment of the present invention, and a conjugate moiety including, but not limited to, a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, the antibody moiety and the conjugate moiety being conjugated via a chemical bond or a linker.
In order to solve the above technical problems, the tenth technical solution of the present invention is: there is provided a pharmaceutical composition comprising an antibody according to one of the claims or a bispecific antibody according to a second of the claims and optionally a pharmaceutically acceptable carrier. Preferably, a pharmaceutically acceptable carrier is also included.
More preferably, the pharmaceutical composition further comprises other anti-tumor antibodies as an active ingredient.
The pharmaceutically acceptable carrier can be a carrier conventional in the art, and the carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical adjuvant is conventional in the field, and preferably comprises pharmaceutically acceptable excipient, filler or diluent and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above antibody and/or the above bispecific antibody, and 0.01-99.99% of a pharmaceutically acceptable carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.
The route of administration of the pharmaceutical composition according to the invention is preferably parenteral, injectable or oral. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is various dosage forms which are conventional in the field, preferably in the form of solid, semisolid or liquid, namely aqueous solution, non-aqueous solution or suspension, and more preferably tablet, capsule, granule, injection or infusion and the like. More preferably via intravascular, subcutaneous, intraperitoneal or intramuscular administration. Preferably, the pharmaceutical composition may also be administered as an aerosol or a coarse spray, i.e. nasally; alternatively, intrathecal, intramedullary or intraventricular administration. More preferably, the pharmaceutical composition may also be administered transdermally, topically, enterally, intravaginally, sublingually or rectally. The pharmaceutical composition of the present invention can be formulated into various dosage forms as required, and can be administered at a dose that is determined by a physician in consideration of the kind, age, weight and general condition of a patient, administration manner, and the like, which are beneficial to the patient. Administration may be by injection or other therapeutic means, for example.
The dosage level of a pharmaceutical composition of the invention administered may be adjusted depending on the amount of the composition to achieve a desired diagnostic or therapeutic result. The administration regimen may also be a single injection or multiple injections, or adjusted. The selected dose level and regimen will be reasonably adjusted depending on various factors including the activity and stability (i.e., half-life) of the pharmaceutical composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and/or treated, and the health and prior medical history of the subject to be treated.
A therapeutically effective dose for the pharmaceutical composition of the invention may be estimated initially in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs and/or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. And can subsequently be used to determine useful doses and routes for administration in humans. In general, the determination and adjustment of the effective amount or dosage to be administered and the assessment of when and how to make such adjustments are known to those skilled in the art.
For combination therapy, the above-described antibody, bispecific antibody, and/or additional therapeutic or diagnostic agent can each be used as a single agent, within any time frame suitable for performing the intended therapy or diagnosis. Thus, these single agents may be administered substantially simultaneously (i.e., as a single formulation or within minutes or hours) or sequentially.
For additional guidance on formulation, dosage, administration regimen, and measurable therapeutic outcomes, see Berkow et al (2000) The Merck Manual of Medical Information (Merck handbook of Medical Information) and Merck & co.inc, Whitehouse Station, New Jersey; ebadi (1998) CRC Desk Reference of Clinical Pharmacology, and the like.
To solve the above technical problems, the eleventh technical solution of the present invention is: there is provided the use of an antibody according to one embodiment of the present invention, a bispecific antibody according to the second embodiment of the present invention, a chimeric antigen receptor according to the third embodiment of the present invention, a genetically modified cell according to the fourth embodiment of the present invention, an antibody drug conjugate according to the ninth embodiment of the present invention or a pharmaceutical composition according to the tenth embodiment of the present invention for the manufacture of a medicament, a kit and/or a delivery device for the treatment and/or prevention of cancer.
Preferably, the cancer is a B7H 4-positively expressed tumor; the tumor is preferably breast cancer, ovarian cancer and endometrioma, and the breast cancer is more preferably triple negative breast cancer.
To solve the above technical problems, the twelfth technical solution of the present invention is: there is provided a method of detecting B7H4 in a sample comprising detecting using an antibody or bispecific antibody as described above. Preferably, the detection method is for non-diagnostic purposes.
Non-diagnostic therapeutic or detection methods of the invention include, but are not limited to: screening of laboratory drugs, preventive medical research and formulation of public health policies, detection using kits, and the like. As known to those skilled in the art, modern medicine is divided into two parts: preventive medicine and clinical medicine. The detection method for non-diagnosis purposes can detect samples (including human body secretion) collected in the environment in preventive medicine and judge whether the environment is polluted by the antigen. In the laboratory, the laboratory reagents can also be tested by the laboratory personnel using the antibody or bispecific antibody of the present invention to ensure that the antigen used in the experiment is not contaminated by antigens other than B7H4, for further use in new antibody screening or screening of small molecule compounds as drug targets, etc.
To solve the above technical problems, the thirteenth technical solution of the present invention is: there is provided a kit comprising an antibody according to one embodiment of the present invention, a bispecific antibody according to a second embodiment of the present invention, a chimeric antigen receptor according to a third embodiment of the present invention, a genetically modified cell according to a fourth embodiment of the present invention, an antibody drug conjugate according to a ninth embodiment of the present invention and/or a pharmaceutical composition according to a tenth embodiment of the present invention, and optionally, instructions.
In order to solve the above technical problems, the fourteenth technical solution of the present invention is: providing a drug delivery device, wherein the drug delivery device comprises: (1) an infusion module for administering a pharmaceutical composition according to the tenth of the present invention to a subject in need thereof, and (2) optionally a pharmacodynamic monitoring module.
To solve the above technical problems, the fifteenth technical solution of the present invention is: there is provided the use of an antibody according to one embodiment of the present invention, a bispecific antibody according to the second embodiment of the present invention, a chimeric antigen receptor according to the third embodiment of the present invention, a genetically modified cell according to the fourth embodiment of the present invention, an antibody drug conjugate according to the ninth embodiment of the present invention, and/or a pharmaceutical composition according to the tenth embodiment of the present invention for the diagnosis, prevention and/or treatment of a tumor. Preferably, the tumor is according to the eighth aspect of the present invention.
To solve the above technical problems, the sixteen technical solutions of the present invention are: there is provided a kit comprising a kit a and a kit B, wherein the kit a is an antibody according to one embodiment of the present invention, a bispecific antibody according to a second embodiment of the present invention, a chimeric antigen receptor according to a third embodiment of the present invention, a genetically modified cell according to a fourth embodiment of the present invention, an antibody drug conjugate according to a ninth embodiment of the present invention, and/or a pharmaceutical composition according to a tenth embodiment of the present invention, and the kit B is another anti-tumor antibody or a pharmaceutical composition comprising the same. The medicine box A and the medicine box B can be used simultaneously, the medicine box A can be used firstly and then the medicine box B can be used, the medicine box B can be used firstly and then the medicine box A can be used, and the medicine box A can be determined according to actual requirements in specific application.
The three letter and one letter codes for amino acids used herein are known to those skilled in the art, or described in j.biol.chem, 243, p3558 (1968). As used herein, the term "comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements but not exclude other elements, but also include the case of "consisting of … …", as the context dictates. The term "or" is used herein to mean and is used interchangeably with the term "and/or" unless the content clearly dictates otherwise. "about" and "approximately" shall generally mean an acceptable degree of error in the measured quantity in view of the nature or accuracy of the measurement. Exemplary degrees of error are typically within 10% thereof and more typically within 5% thereof and even within 2% or 1% thereof. As used herein, the term EC50Means the half-maximal effect concentration (concentration for 50% of the maximum effect), i.e. the concentration that causes 50% of the maximal effect.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
1. the B7H 4-targeting monoclonal antibody of the invention is a naturally occurring fully human antibody with binding activity to human B7H4 and cynomolgus monkey B7H4, without cross-reactivity with other B7 family members. After Fc modification, the antibody has stronger ADCC effect, and the in vivo experiment shows good antitumor activity. PR003369 has higher T cell activation activity and higher internalization activity after affinity maturation modification, and is more suitable for serving as an ADC (azodicarbonamide) medicament.
2. The B7H4 XCD 3 bispecific antibody has a bispecific antibody structure with a human Fc fragment, retains the binding effect of Fc and FcRn, and has longer half-life. The end of B7H4 adopts ScFv form, which simplifies the mismatch of light and heavy chains and retains good stability and hydrophilicity. Optimizes the activity of CD3 terminal, adopts medium strength anti-CD 3 antibody, reduces toxicity on the premise of ensuring drug effect. The B7H4 terminal and CD3 terminal antibodies have good binding activity to cynomolgus monkeys.
Drawings
FIG. 1 is the result of binding of the primary antibody to human B7H4 on the cell surface;
FIG. 2 is the results of PR001476 and PR002037 and their PTM variant/DE mutant antibodies expressing supernatant binding to human B7H4 on the cell surface;
FIG. 3 is the results of binding of primary antibody to cynomolgus monkey B7H4 on the cell surface;
FIG. 4 is the results of PR001476 binding to cynomolgus monkey B7H4 on the cell surface with PR002037 and its PTM variant/DE mutant antibody;
FIG. 5 is the results of initial antibody binding to cell surface mouse B7H 4;
FIG. 6 is the results of PR001476 and PR002037 and its PTM variant/DE mutant antibody binding to mouse B7H4 at the cell surface;
FIG. 7 shows the results of binding of primary antibodies to B7H4 on the surface of tumor cells SK-BR-3;
FIG. 8 is the result of PR001476 binding to B7H4 on the surface of tumor cells SK-BR-3 with PR002037 and its PTM variant/DE mutant antibody supernatant;
FIG. 9 shows the results of PR001476 binding to B7H4 on the surface of tumor cells SK-BR-3 with PR002037 and its PTM variant/DE mutant antibody;
FIG. 10 shows the result that PR002418 and PR001476 affinity maturation/DE mutant antibody PR003369 bind to B7H4 on the surface of tumor cells MDA-MB-468;
FIG. 11 is ADCC killing activity of PR001476 and its PTM variant/DE mutant antibody against SK-BR-3 tumor cells;
FIG. 12 is the ADCC killing activity of PR002037 and its PTM variant/DE mutant antibodies against SK-BR-3 tumor cells;
FIG. 13 is a comparison of ADCC killing activity of PR002418, PR002421 against SK-BR-3 tumor cells with control antibodies from FivePrime;
FIG. 14 compares the ADCC killing activity of PR003369, PR002418, RP002421 against MDA-MB-468, HCC-1954 tumor cells with control antibody 2 from FivePrime;
FIG. 15 is a result of the anti-B7H4 antibody blocking the immunosuppressive signal of B7H4 to activate T cells;
FIG. 16 is the result of internalization of anti-B7H4 antibody on SK-BR-3 cells;
FIG. 17 is activity of anti-B7H4 antibody internalization mediated cytotoxicity;
FIG. 18 is a graph of the effect of MMAE group cross-linking on the B7H4 binding activity of antibodies on tumor cell surfaces;
FIG. 19 is a graph of the effect of MMAE group cross-linking on the internalization activity of antibodies on tumor cells;
FIG. 20 shows the tumor cell killing efficiency of the affinity matured variant PR003369-ADC and the control antibody PR 000157-ADC;
FIG. 21 is an analysis of antibody affinity data by Biacore T200 analysis software 2.0;
FIG. 22 is a cross-reactive representation of anti-B7H4 antibody with other members of the B7 family;
FIG. 23 is a graph of the stability of anti-B7H4 antibodies PR002418, PR002037, PR003369 and control antibodies in 37 ℃ human serum for 14 days;
figure 24 is a graph of the half-lives of PR002418 and PR002421 and control antibody 2 in mice;
FIG. 25 shows the in vivo anti-tumor effect of anti-B7H4 monoclonal antibody molecules in BALB/c nude mouse MDA-MB-468 tumor model;
FIG. 26 is a graph of the in vivo anti-tumor effect of anti-B7H4 monoclonal antibody molecules in an MDA-MB-468 tumor model of NSG mice reconstitution of human PBMC immune system;
FIG. 27 is B7H4 expression in normal tissue (A) and tumor (B) and its IHC score statistics (C);
FIG. 28 is a schematic diagram of the structure of the B7H4 XCD 3 diabody molecule;
FIG. 29 is the results of B7H4 XCD 3 diabodies binding to SK-BR-3 cells (A-D) and T cells (E-H);
FIG. 30 shows the results of B7H4 XCD 3 dual antibody (A-L, "1 + 1" asymmetric structure; M-N, "2 + 1" asymmetric structure) activating T cells and killing target cells;
FIG. 31 shows the tumor suppression rates of B7H4 XCD 3 double antibody in a tumor model (A, MDA-MB-468 model; B, HCC-1954 model) in NSG mice to reconstitute the human PBMC immune system.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.
Example 1 acquisition of anti-B7H4 antibody molecule
Experimental animals, which may be mice, rats, rabbits, sheep, camels, etc., may be immunized with B7H4 recombinant protein or cells overexpressing B7H4 to obtain antibody molecules that specifically bind to B7H 4. Typically, the resulting antibody molecules are of non-human origin. After obtaining non-human antibodies, these molecules need to be humanized using antibody engineering techniques to reduce immunogenicity and improve druggability. However, the humanization process of antibodies has technical complexity, and molecules that are humanized and modified tend to have reduced affinity for antigens. On the other hand, advances in transgenic technology have made it possible to develop genetically engineered mice that carry the human immunoglobulin immune repertoire and have the endogenous murine immune repertoire deleted. The Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying human immunoglobulin immune repertoire, and the antibody generated by the transgenic mouse has a fully human sequence, so that further humanized modification is not needed, and the efficiency of developing therapeutic antibody is greatly improved.
1.1. Immunization of mice
Harbour H2L2 mice were subjected to multiple rounds of immunization with soluble recombinant human B7H4-ECD-mFc fusion protein (Sino Biological, #10738-H05H) as antigen. The antigenic protein is mixed with an immunological adjuvant to form an immunogen reagent, and then the immunogen is injected subcutaneously through the groin or is injected intraperitoneally. In each round of immunization, each mouse received a total injection dose of 100 microliters. In the first round of immunization, each mouse received an immunization with 50 micrograms of antigenic protein mixed with complete Freund's adjuvant (Sigma, # F5881) in a 1: 1 volume ratio of the immunogenic reagent. In each subsequent round of booster immunizations, each mouse received an immunization with an immunogenic reagent formulated with 25 micrograms of antigenic protein mixed with Sigma Adjuvant System Adjuvant (Sigma, # S6322). The interval between each round of booster immunization is at least two weeks, usually 6 to 7 rounds of booster immunization. The immunization time is 0, 14, 28, 42, 56, 70, 84 and 98 days; and at days 49, 77, mouse serum antibody titers were detected. The last booster immunization was performed 5 days before H2L2 mouse splenic B cell isolation, at a dose of 25 micrograms of antigenic protein per mouse.
Or transfecting CHO-K1 cell (CHO-K1/hu B7H4 and platinum medicine) for over-expressing human B7H4 with plasmid encoding mouse CD40L, mixing with immune adjuvant to obtain immunogen reagent, and immunizing mice with 5 × 10 cells per mouse6The cell and immune process are immune with protein.
1.2. Serum titer detection
At specific time points, mouse sera were harvested and the titers of antibody binding to B7H4 protein in the sera were measured by ELISA and the titers of antibody binding to B7H 4-overexpressing cells in the sera were measured by FACS.
In the ELISA method, 100. mu.L/well of hB7H4-ECD-his protein (Sino Biological, #10738-H08H) was coated on an ELISA plate (corning, 9018) at 1. mu.g/mL and incubated overnight at 4 ℃; after 2 rinses, blocking with 1% BSA in PBST for 2 hours at 37 ℃; adding 100 μ L/well diluted serum and incubating at 37 deg.C for 1 hr; after 3 rinses, 100. mu.L/well of 1: 5000 diluted anti-rat-HRP (sigma, # A5795) was added and incubated at 37 ℃ for 30 minutes. After 3 rinses, 100. mu.L/well TMB substrate was added and incubated for about 10 minutes, and the development was stopped by adding 50. mu.L/well 1N HCl and the absorbance at 450nm was read (Molecular Devices, Plus 384).
In the FACS method, gradient diluted murine serum was incubated with HEK293-B7H4 cells for 1 hour at 4 ℃; after 2 washes of the cells, a secondary anti-rat IgG (H + L) (Life technologies, A11006) was added and incubated at 4 ℃ for 1 hour, after 2 washes, the resuspended cells were detected with a flow cytometer (BD, Flibur). HEK293 cells served as background controls.
1.3. Screening of antibodies against B7H4 by hybridoma technology
Selecting immune mice with high serum titer, carrying out primary final immunization, killing the mice, taking spleen cells and SP2/0 myeloma cells (ATCC, CRL-1581) for electrofusion, wherein the cell ratio is 4: 1, and the electrofusion parameters are V1: 50V, t 1: 15s, V2: 600V, t 2: 20 μ s, t 3: 0.5s, n: 1, t 4: 7s, V +/-: +, fade: and on. Cells were resuspended in culture medium of DMEM containing 20% FBS and HT in 1X 10 plates5At 100. mu.L/well, 24 hours later, 100. mu.L/well of DMEM containing 20% FBS and 2 XHT was added to continue the culture. Subsequently, the supernatant was removed to detect the antibody titer. Generally, at 9-15 days after fusion, supernatants from protein immunized mice were primary screened with Acumen to detect binding to CHO-K1/huB7H4 cells; cell-immunized mice supernatants were collected and purified by Mirrorball (SPT Labtech,
Figure PCTCN2021102952-APPB-000001
fluoroscience cytometer) to detect binding to HEK-293/huB7H4 cells. Positive clones were then further confirmed by ELISA and FACS to examine the binding ability to CHO-K1 cell lines overexpressing human B7H4 (CHO-K1/huB7H4), CHO-K1 cell lines overexpressing cynomolgus monkey B7H4 (CHO-K1/cynoB7H4), CHO-K1 cell lines overexpressing mouse B7H4 (CHO-K1/mB7H 4). Positive wells were further subcloned by limiting dilution and further screened by ELISA and FACS methods. Clones that bound well to human and monkey B7H4 were selected for sequencing.
1.4. Screening of anti-B7H4 antibodies by B cell in vitro cloning technology
The spleens of the mice were removed, ground and filtered through a 200 mesh filter, and the single cell suspensions were sorted according to the mouse memory B cell sorting kit (Miltenyi, # 130-. And (4) carrying out immunofluorescence staining on the sorted cells.
B200 positive (BioLegend, #103227), IgM negative (BioLegend, #406506), B7H4 specific positive cells (BioLegend, #405207) were sorted on a flow cytometer S3 e. The cells obtained by sorting were cultured in a cell culture 96-well plate at a density of 5 cells per well, and irradiated EL4 cells were previously plated on the cell culture plate as feeder cells.
After 14 days of culture, culture supernatants were collected and assayed by ELISA, and for wells having binding activity to B7H4 protein, cells were removed and subjected to RT-PCR (SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (#634892), I-5TM2 × High-Fidelity Master Mix (# I5 HM-5000). Splicing the light chain and the heavy chain obtained by amplification into scFv by overlap PCR, expressing in E.coli, carrying out ELISA detection on the expression supernatant, and sequencing the positive clone.
1.5. Sequence analysis and sequence optimization of anti-B7H4 antibodies
The nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence are obtained using conventional sequencing means. 3 monoclonal sequences were obtained. In this example, the sequence of the anti-B7H4 monoclonal antibody molecule variable domain obtained from immunized Harbour H2L2 mice was a human antibody sequence, and the germline gene analysis and post-translational modification site (PTM) analysis thereof are listed in Table 1-1.
Chemical modifications, known as post-translational modifications (PTMs), are sometimes introduced after the translational synthesis of a protein or polypeptide amino acid chain in a cell. For antibodies, the site of some PTMs is very conserved, e.g., the conserved amino acid asparagine Asn at position 297 (EU numbering) of the constant domain of the human IgG1 antibody is usually glycosylated to form a sugar chain whose structure is critical for antibody structure and associated effector functions. However, if PTMs are present in the variable domains, particularly the antigen binding regions (e.g., CDRs), of an antibody, then the presence of these PTMs may have a greater effect on antigen binding and may also have a change in the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, oxidation, and the like may all increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development. Avoiding some potential PTMs is therefore very important for the development of therapeutic antibodies. As experience has accumulated, it has been found that some PTMs are highly correlated with the composition of amino acid sequences, particularly the "pattern" of adjacent amino acid compositions, such that potential PTMs can be predicted from the primary amino acid sequence of the protein. For example, the sequence pattern of N-x-S/T (asparagine at the first position, any amino acid other than non-proline at the second position, and serine or threonine at the third position) predicts an N-linked glycosylation site. It is possible that the amino acid sequence pattern leading to PTM is derived from an germline gene sequence, e.g.the human germline gene segment IGHV3-33 naturally has a glycosylation pattern NST in the FR3 region; it may also be derived from somatic high-frequency mutations.
The amino acid sequence pattern of a PTM may be disrupted by amino acid mutation, thereby reducing or eliminating the formation of a particular PTM. There are different mutation design methods depending on the antibody sequence and PTM sequence pattern. One approach is to replace the "hot spot" amino acid (e.g., N or S in the NS pattern) with an amino acid with similar physicochemical properties (e.g., mutation of N to Q). If the PTM sequence pattern is derived from somatic high-frequency mutations and is not present in the germline gene sequence, an alternative would be to replace the sequence pattern with the corresponding germline gene sequence. In practice, multiple mutation design methods may be used for the same PTM sequence pattern.
Tables 1-2 list the sequences of novel antibody molecules obtained by amino acid mutation of the sequences of antibodies PR001476 and PR 002037.
TABLE 1-1 germline gene analysis and PTM site analysis of anti-B7H4 antibodies
Figure PCTCN2021102952-APPB-000002
TABLE 1-2 mutant site design of antigen binding protein sequences
Figure PCTCN2021102952-APPB-000003
PR001476 antibody affinity maturation
The molecule PR001476 was engineered by site-directed mutagenesis to increase its affinity for binding to B7H 4. This method of affinity maturation is divided into two rounds.
In the first round, amino acids of the heavy chain CDR3 and light chain CDR3 (defined as Chothia CDRs) of PR001476 molecule were scanned point-by-point to create a single point saturated mutant library of multiple amino acid positions. And (3) screening the 2 saturated mutation library, picking out positive molecules with signals 2 times more than those of wild type for sequencing, further identifying the positive molecules, and selecting a plurality of mutation hot spots according to the binding capacity of the positive molecules to human B7H 4.
In the second round, the hot spots found by the saturated mutations in the first round are randomly combined to establish a library containing all mutation combinations. The combinatorial library is then screened. Positive molecules several mutants were selected by sequencing and binding to human hB7H 4. The selected mutants are indicated by the corresponding clone numbers, for example: PR001476-R1-25B3, PR001476-R1-26D7, and the like.
The mutant is constructed into a mammalian expression vector for expression and purification of the protein. The mutants were then tested for their ability to bind to B7H4 using FACS and Fortebio Octet. PR003369 in tables 1-2 is a preferred mutant derived from PR 001476.
1.7. Recombinant antibody preparation and physicochemical property characterization analysis
1.7.1. Expression and purification of antibodies
This example describes a general method for producing antibodies using mammalian host cells (e.g., human embryonic kidney cell HEK293 or Chinese hamster ovary cell CHO and derived cells), transient transfection expression, and affinity capture isolation. The method is applicable to target antibodies containing an Fc region; the target antibody may be composed of one or more protein polypeptide chains; may be derived from one or more expression plasmids.
Converting the amino acid sequence of the antibody polypeptide chain into a nucleotide sequence by a codon optimization method; the encoded nucleotide sequence is synthesized and cloned into an expression vector compatible with the host cell. Transfecting the plasmid encoding the polypeptide chain of the antibody into a mammalian host cell simultaneously according to a specific ratio, and obtaining the polypeptide chain of the antibody by using a conventional recombinant protein expression and purification technologyTo recombinant antibodies with proper folding and polypeptide chain assembly. In particular, FreeStyleTM293-F cells (Thermo, # R79007) in FreeStyleTMF17 Expression Medium (Thermo, # A1383504). Prior to the start of transient transfection, cell concentrations were adjusted to 6-8X 105cells/mL, 8% CO at 37 ℃2Culturing in shaking bed for 24 hr to obtain cell concentration of 1.2 × 106cells/mL. 30mL of cultured cells were prepared. Plasmids encoding the polypeptide chains of the antibody were mixed in a ratio such that 30. mu.g of plasmid (plasmid to cell ratio 1. mu.g: 1mL) was dissolved in 1.5mL of Opti-MEM serum-reduced medium (Thermo, #31985088) and sterilized by filtration through a 0.22 μm filter. Then, 120. mu.l of 1mg/mL PEI (Polysciences, #23966-2) was dissolved in 1.5mL Opti-MEM and allowed to stand for 5 minutes. Slowly adding PEI into the plasmid, incubating for 10 min at room temperature, slowly dropping the mixed solution of the plasmid PEI while shaking the culture flask, and adding 8% CO at 37 deg.C2Cultured in a shaker for 5 days. Cell viability was observed after 5 days. Collecting the culture, centrifuging at 3300g for 10 min, and collecting the supernatant; the supernatant was then centrifuged at high speed to remove impurities. Equilibration of the buffer containing MabSelect with PBS pH7.4TM(GE Healthcare, #71-5020-91) gravity column (Bio-Rad, #7311550), 2-5 column volumes washed. Passing the supernatant sample through a column; the column was washed with 5 to 10 column volumes of PBS buffer, followed by elution of the protein of interest with 0.1M glycine at pH3.5, followed by adjustment to neutrality with Tris-HCl pH 8.0, and finally by concentration of the exchange solution to PBS buffer or a buffer containing other components using an ultrafiltration tube (Millipore, # UFC901024) to obtain a purified recombinant antibody solution. Finally using NanoDrop (Thermo, NanoDrop)TMOne) measuring the concentration, subpackaging and storing for later use.
1.7.2. Protein purity and aggregation analysis by SEC-HPLC
This example uses analytical Size Exclusion Chromatography (SEC) to analyze protein samples for purity and for the form of aggregates. Analytical column TSKgel G3000SWX1(Tosoh Bioscience, #08541, 5 μm, 7.8 mm. times.30 cm) was attached to a high pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 μ g) was filtered through a 0.22 μm filter and injected into the system and the HPLC program was set: the sample was flowed through the column with PBS buffer at a flow rate of 1.0 mL/min for a maximum of 25 minutes. HPLC will generate an analytical report reporting the residence time of different molecular size components within the sample.
1.7.3. Protein purity and hydrophobicity analysis by HIC-HPLC
Analytical Hydrophobic Interaction Chromatography (HIC) was used to analyze protein samples for purity and hydrophobicity. Analytical column TSKge1 Buty1-NPR (Tosoh Bioscience, 14947, 4.6 mm. times.3.5 cm) was connected to high pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. The procedure was set up from a linear gradient from 100% mobile phase A (20mM histidine, 1.8M ammonium sulfate, pH 6.0) to 100% mobile phase B (20mM histidine, pH 6.0) in 16 minutes, with a flow rate set at 0.7 mL/min, a protein sample concentration of 1mg/mL, a sample volume of 20. mu.l, and a detection wavelength of 280 nm. And integrating the chromatogram by using ChemStation software after collection, calculating relevant data, generating an analysis report, and reporting the retention time of components with different molecular sizes in the sample.
1.7.4. Determination of the thermal stability of protein molecules using DSF
Differential Scanning Fluorescence (DSF) is a commonly used high-throughput method for determining the thermal stability of proteins. It uses real-time fluorescent quantitative PCR instrument to reflect the denaturation process of protein by monitoring the change of fluorescence intensity of dye combined with unfolded protein molecule, thus reflecting the thermal stability of protein molecule. This example uses the DSF method to determine the thermal denaturation temperature (Tm) of protein molecules. Mu.g of protein was added to a 96-well PCR plate (Thermo, # AB-0700/W), followed by 2. mu.l of 100 Xdilution dye SYPROTM (Invitrogen, #2008138), and then buffer was added to give a final volume of 40. mu.l per well. The PCR plate was sealed, placed in a real-time fluorescent quantitative PCR instrument (Bio-Rad CFX96 PCR System), incubated at 25 ℃ for 5 minutes, then gradually warmed from 25 ℃ to 95 ℃ with a gradient of 0.2 ℃/0.2 minutes, and at the end of the test the temperature was lowered to 25 ℃. Data analysis and calculation of Tm for samples were performed using FRET scanning mode and Bio-Rad CFX Maestro software.
1.8. Preparation of anti-B7H4 fully human recombinant antibody
The fully human IgG antibodies against B7H4 obtained in 1.3-1.6 and the optimized antibodies were prepared and analyzed as described in 1.7.1. Tables 1-3 and tables 1-4 list the results of small and large volume transient expression purification, respectively. In addition, anti-B7H4 antibody sequences (tables 1-5) were obtained from the literature and used as controls in subsequent experiments.
TABLE 1-3 expression of anti-B7H4 antibodies
Antibodies Expression System and volume Yield after one-step purification (mg/L) SEC-HPLC purity (%)
PR001476 HEK293-F(50mL) 80.6 99.18
PR002037 HEK293-F(30mL) 17.3 100
PR002038 HEK293-F(30mL) 34.0 100
PR002408 HEK293-6E(40mL) 37.0 99.03
PR002411 HEK293-6E(40mL) 64.8 99.3
PR002418 HEK293-F(1.5mL) 26.7 Not testing
PR003369 HEK293-F(30mL) 6.6 100
PR002410 HEK293-6E(40mL) 173.5 98.65
PR002420 HEK293-6E(40mL) 5.7 99.16
PR002421 HEK293-F(1.5mL) 73.3 Not testing
TABLE 1-4 expression of anti-B7H4 antibodies
Figure PCTCN2021102952-APPB-000004
Tables 1-5 information relating to control antibodies
Figure PCTCN2021102952-APPB-000005
1.9. anti-B7H4 antibody sequences and numbering
In the present invention, the amino acid sequences of the above-listed CDRs are shown according to the Chothia definition rules. However, it is well known to those skilled in the art that the CDRs of an antibody can be defined in the art by a variety of methods, such as the Kabat definition rule based on sequence variability (see Kabat et al, immunological protein sequences, fifth edition, national institutes of health, Besserda, Md. (1991)) and the Chothia definition rule based on the position of the structural loop region (see Jmol Biol 273: 927-48, 1997). In the context of the present invention, the rules of the Combined definition, including the Kabat definition and the Chothia definition, may also be used to determine amino acid residues in variable domain sequences. The Combined definition rule combines the ranges defined by Kabat and Chothia, and takes a larger range based on the Combined definition rule, which is shown in tables 1-6. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) will be understood to encompass complementarity determining regions as defined by any of the above known schemes described by this invention. Although the scope of the invention is claimed based on the sequence shown in the Chothia definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of the invention.
Tables 1-6 methods for CDR definition in antibodies of the present application
Kabat Chothia Combined
LCDR1 L24--L34 L24--L34 L24-L34
LCDR2 L50--L56 L50--L56 L50-L56
LCDR3 L89--L97 L89--L97 L89-L97
HCDR1 H31--H35 H26--H32 H26-H35
HCDR2 H50--H65 H52--H56 H50-H65
HCDR3 H95--H102 H95--H102 H95-H102
Wherein Laa-Lbb can refer to the amino acid sequence from position aa (Chothia coding rule) to position bb (Chothia coding rule) from the N-terminus of the antibody light chain; Haa-Hbb can refer to the amino acid sequence from position aa (Chothia coding rule) to position bb (Chothia coding rule) from the N-terminus of the antibody heavy chain. For example, L24-L34 may refer to the amino acid sequence from position 24 to position 34, beginning at the N-terminus of the antibody light chain, according to the Chothia coding rules; H26-H35 can refer to the amino acid sequence from position 26 to 35 beginning at the N-terminus of the antibody heavy chain according to the Chothia coding rules. It will be appreciated by those skilled in the art that there are positions where insertion sites are present when encoding a CDR using Chothia (see httD:// bioif. org. uk/abs /).
Tables 1-7 list the sequence numbers of the CDRs, variable regions and light and heavy chains corresponding to the sequences of the anti-B7H4 antibodies of the invention and the control antibody molecules. PR003366 is a single chain variable region (scFv) homodimer molecule (scFv-Fc structure) constructed using the variable region sequence of PR 002410.
Tables 1-7 sequence numbering of anti-B7H4 antibodies
Antibody numbering Light chain Heavy chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
PR000157 108 94 85 75 45 52 62 7 19 33
PR002961 116 104 92 82 48 54 68 12 25 37
PR002962 116 105 92 82 48 54 68 12 25 37
PR001476 109 95 87 77 47 54 64 9 21 35
PR002037 112 98 88 79 48 54 65 11 22 36
PR002038 113 98 89 79 48 55 66 11 22 36
PR002405 114 99 90 80 47 54 67 9 23 35
PR002408 114 100 90 81 47 54 67 9 24 35
PR002411 109 101 87 77 47 54 64 9 21 35
PR002418 114 102 90 81 47 54 67 9 24 35
PR003369 117 106 93 83 47 54 69 9 24 38
PR002410 115 98 91 79 48 54 65 11 22 36
PR002420 112 103 88 79 48 54 65 11 22 36
PR002421 115 103 91 79 48 54 65 11 22 36
PR003366 132 91 79 48 54 65 11 22 36
Tables 1-8 list the sequence numbers of the framework regions, Fv, corresponding to the sequences of the anti-B7H4 antibodies of the invention and the control antibody molecule.
TABLE 1-8 framework regions and Fv sequence numbering for anti-B7H4 antibodies
Figure PCTCN2021102952-APPB-000006
Figure PCTCN2021102952-APPB-000007
Tables 1-9 list the CDR sequences corresponding to the sequences of the anti-B7H4 antibodies of the present invention and the control antibody molecules.
TABLE 1-9 CDR sequences of anti-B7H4 antibodies
Figure PCTCN2021102952-APPB-000008
Example 2 FACS detection of the ability of anti-B7H4 antibodies to bind to B7H4
This example is to investigate the in vitro binding activity of H2L2 mab against human B7H4 to human/cynomolgus monkey/mouse B7H 4. CHOK1 cell line (CHOK1/hu B7H4 and platinum medicine) over-expressing human B7H4, CHOK1 cell line (CHOK1/cyno B7H4 and platinum medicine) over-expressing cynomolgus monkey B7H4, CHOK1 cell line (CHOK1/mB7H4 and platinum medicine) over-expressing mouse B7H4 and cell line SK-BR-3 (CHOK1/mB7H 4) highly expressing human B7H4 are used
Figure PCTCN2021102952-APPB-000009
HTB-30) were subjected to antibody binding experiments at the cellular level. Briefly, CHOK1/huB7H4 cells, CHOK1/cyno B7H4 cells, CHOK1/m B7H4 cells, or SK-BR-3 cells were digested and resuspended in PBS containing 2% BSA. Will be provided withCell densities were adjusted to 1X 106cells/mL. Cells were seeded at 100. mu.L/well in 96-well V-plates (Corning, #3894), followed by the addition of 100. mu.L/well of test antibody diluted in a 3-fold concentration gradient 2-fold the final concentration. Cells were incubated at 4 ℃ for 2 hours in the dark. Thereafter, cells were rinsed twice with 100. mu.L/well of pre-chilled 2% BSA in PBS, centrifuged at 500g for 5 minutes at 4 ℃ and the supernatant discarded. Then 100. mu.L/well fluorescent secondary antibody (AlexaFluor488-conjugated affinity assay Goat Anti-Human IgG, Fc gamma Fragment specificity, Jackson, # 109-. Cells were washed twice with 100. mu.L/well pre-chilled 2% BSA in PBS, centrifuged at 500g for 5 min at 4 ℃ and the supernatant discarded. Finally, cells were resuspended in 200. mu.L/well pre-cooled 2% BSA in PBS and the fluorescence emission signal was read using an ACEA Novocyte3000 flow cytometer.
A summary of antibody binding to cell surface human B7H4, cynomolgus monkey B7H4 and mouse B7H4, and to tumor cell SK-BR-3 surface B7H4 is as follows (Table 2-1, Table 2-2, Table 2-3). The affinity matured variant PR003369 showed a marked increase in the binding to tumor cells compared with PR002418 (tables 2 to 3).
TABLE 2-1 anti-B7H4 antibody (Primary antibody) binds to human B7H4, cynomolgus monkey B7H4, mouse B7H4 and B7H4 bound to the surface of tumor cells SK-BR-3
Figure PCTCN2021102952-APPB-000010
TABLE 2-2 anti-B7H4 antibodies (variant molecules) bind to cynomolgus monkey B7H4, mouse B7H4 on the cell surface and B7H4 on the surface of tumor cells SK-BR-3
Figure PCTCN2021102952-APPB-000011
Figure PCTCN2021102952-APPB-000012
TABLE 2-3 binding of PR003369 and PR002418 to tumor cells
Figure PCTCN2021102952-APPB-000013
The results of the initial antibody binding to human B7H4 on the cell surface are shown in FIG. 1, and the results show that PR001476 and PR002037 have good human B7H4 binding activity and poor PR002038 binding activity; the results of PR001476 binding to PR002037 and its PTM variant/DE mutation the results of the supernatant binding to human B7H4 on the cell surface are shown in fig. 2, showing that the PTM variant/DE mutation of PR002037, PR001476 did not significantly affect the binding activity of the antibody to human B7H 4. The results of binding of the initial antibody to cynomolgus monkey B7H4 on the cell surface are shown in fig. 3, and the results show that PR002037 and PR001476 have better cross-binding activity of cynomolgus monkey B7H4 and poorer cross-binding activity of PR 002038; the results of PR001476 binding to cell surface cynomolgus monkey B7H4 with PR002037 and its PTM variant/DE mutant antibodies are shown in fig. 4, showing that the PTM variant/DE mutations of PR002037, PR001476 did not significantly affect the cross-binding activity of the antibodies with cynomolgus monkey B7H 4. The results of initial antibody binding to cell surface mouse B7H4 are shown in fig. 5, and show that PR002037 has better mouse B7H4 cross-binding activity, PR001476 has weaker mouse B7H4 cross-binding activity, and PR002038 has no mouse B7H4 cross-binding activity; the results of PR001476 binding to cell surface mouse B7H4 with PR002037 and its PTM variant/DE mutant antibody are shown in fig. 6, showing that the PTM variant/DE mutant antibodies of PR002037, PR001476 retained similar cross-binding activity of mouse B7H4 compared to their parent. The result of the initial antibody binding to B7H4 on the surface of the tumor cell SK-BR-3 is shown in FIG. 7, and the result shows that PR002037 and PR001476 have better binding activity with B7H4 on the surface of the tumor cell SK-BR-3 and have poorer binding activity with PR 002038; the results of PR001476 binding to B7H4 on the surface of tumor cells SK-BR-3 with PR002037 and its PTM variant/DE mutant antibody supernatant are shown in fig. 8, which shows that the PTM variant/DE mutation of PR002037, PR001476 does not significantly affect the binding activity of the antibody to B7H4 on the surface of tumor cells SK-BR-3; the results of PR001476 binding to B7H4 on the surface of tumor cells SK-BR-3 with PR002037 and its PTM variant/DE mutant antibody are shown in FIG. 9, and the results show that the PTM variant/DE mutant antibodies of PR002037 and PR001476 retain the binding activity to B7H4 on the surface of tumor cells SK-BR-3. Wherein the PTM variant/DE mutant antibody of PR001476 had a stronger binding activity (lower EC50) than control antibody 1; the results of the affinity maturation/DE mutant antibody PR003369 binding to B7H4 on the surface of tumor cells MDA-MB-468 for PR002418 and PR001476 are shown in fig. 10, which shows a clear increase in the binding of the affinity maturation variant PR003369 to B7H4 on the surface of tumor cells MDA-MB-468 compared to PTM variant PR 002418.
Example 3 ADCC Activity assay
This example is intended to investigate the activity of H2L2 mAb against human B7H4 in vitro in mediating NK cell killing of target cells by ADCC effect. Human PBMC is used as effector cells, and cell lines SK-BR-3 and MDA-MB-468 highly expressing B7H4 and cell line HCC-1954 expressing B7H4 are used as target cells. The killing efficiency was reflected by the detection of the conductivity of the target cells using the ACEA RTCA instrument. The 96-well plate e-plate was first equilibrated with 50. mu.l of complete medium. SK-BR-3, MDA-MB-468 or HCC-1954 cells were digested, resuspended in RPM1640 complete medium containing 10% fetal bovine serum, and diluted to 4X 105mL, 50. mu.l/well in e-plate 96 plates, i.e.2X 104Perwell, incubated overnight at 37 ℃. The next day, fresh 50. mu.l of 2X 10 solution was added to each well5PBMC culture solution, then add 50 u l4 x concentration gradient dilution of antibody, antibody maximum final concentration of 10nM, each antibody total 8 concentrations, set up two repeat. The conductivity of the target cells was measured in real time and the target cell killing efficiency was calculated as (1-sample/blank) × 100%, typically taking the data at the 4 hour time point.
The ADCC killing activity of PR001476 and PTM variant/DE mutant antibody on SK-BR-3 tumor cells is shown in figure 11, and the result shows that the DE mutation (PR002418) at the Fc end can obviously enhance the ADCC killing activity of the antibody on the SK-BR-3 tumor cells; the ADCC killing activity of PR002037 and PTM variant/DE mutant antibody on SK-BR-3 tumor cells is shown in figure 12, and the result shows that the DE mutation (PR002421) at the Fc end can obviously enhance the ADCC killing activity of the antibody on the SK-BR-3 tumor cells. Comparison of ADCC killing activity of PR002418, PR002421 against SK-BR-3 tumor cells with control antibodies from veprime is shown in fig. 13 (where a is donor 1 and B is donor 2 data), and the results show that PR002418 has similar ADCC killing activity against SK-BR-3 tumor cells compared to the veprime control antibody, and PR002421 killing activity is slightly weaker. PR003369, PR002418 and RP002421 were compared with control antibody 2 from FivePrime on MDA-MB-468 and HCC-1954 tumor cells in ADCC killing activity (wherein A is MDA-MB-468, and B is HCC-1954 data) in FIG. 14, and the results showed that PR003369, PR002418 and RP002421 all have ADCC killing activity on MDA-MB-468 and HCC-1954 tumor cells, and the killing activity is positively correlated with B7H4 expression, i.e. the killing activity is stronger on MDA-MB-468 cells with high expression of B7H4, and weaker on HCC-1954 cells with expression of B7H 4. The affinity matured variant PR003369 further enhanced ADCC killing activity compared to PR002418, PR 002421.
Example 4 detection of T cell activation Activity
In order to detect the function of anti-B7H4 antibody in blocking T cell immune checkpoint and thus activating T cells, the experiment overexpressed full-length B7H4 on HEK293T cells and anti-human CD3 antibody OKT3 in scFv form as artificial antigen presenting cells (HEK293T/OS8/hB7H4, Beijing Kangyuan), separated T cells using human T cell isolation kit (Miltenyi, #130-096-535) according to the instructions, co-cultured the artificial antigen presenting cells with T cells, and tested the effect of anti-B7H4 antibody on T cell activation. Specifically, HEK293T-OS8-hB7H4 was expressed as 1X 104Density plated per well and incubated overnight. Isolation of human Primary T cells at 2X 105A density of 100. mu.l/well was added to HEK293T/OS8/hB7H4 cells. Two replicates were then set up by adding 100 μ L/well of test antibody diluted in a 5-fold concentration gradient of 2 x final concentration, with the highest final concentration of antibody being 10nM, for 6 concentrations of each antibody. After 3 days of culture, the supernatant was collected and the IFN-. gamma.concentration was measured by ELISA. The results show that PR003369, PR002418, PR002421 and the control antibody can promote T cell activation, wherein the affinity matured variant PR003369 has stronger T cell activation activity than the PTM variant PR002418 and the control antibody 2, and the effect is that the affinity matured variant PR003369 has stronger T cell activation activityThe mechanism may be to block the interaction between B7H4 and its unknown receptor on T cells. The results of the anti-B7H4 antibody blocking the immunosuppressive signal of B7H4 to activate T cells are shown in fig. 15 (where a is donor 1 and B is donor 2).
Example 5 antibody internalization experiments
Internalization of the antibody was detected using the Zenonenphrodo iFL IgG Labeling Reagents kit (Invitrogen, # Z25611). The reagent is a secondary antibody with a fluorescent dye, does not emit fluorescence at neutral pH, can automatically emit bright fluorescence in an acidic pH environment after being combined with the primary antibody and internalized into lysosomes along with the antibody, and can be detected by a FACS method. The specific method comprises the following steps: SK-BR-3 cells were collected, centrifuged, supernatant was discarded, cells were suspended in medium, and cell concentration was adjusted to 3X 106and/mL. The cell suspension was added to a 96-well plate at 50. mu.l/well, and then incubated overnight in a 37 ℃ incubator. 4X test antibody was prepared at a maximum concentration of 40nM (4X), 3-fold dilutions, for a total of 8 dilutions. A4 XZenon solution was prepared, and 25. mu.l of the antibody to be tested and 25. mu.l of the 4 XZenon labeling solution were mixed together and left at room temperature for 5 minutes. Then 50. mu.l of the labeled antibody was added to a 96-well plate containing cells, and incubated in a 37 ℃ incubator for 24 hours. The cells were digested and fluorescence read on a flow cytometer. The results show that PR003369 has the highest internalization activity compared to the control antibody RP000014 and other antibodies. Results of internalization of anti-B7H4 antibodies on SK-BR-3 cells are shown in FIG. 16.
Internalization of the antibody was detected using a-HFc-CL-MMAF reagent (Moradec, # AH-102-AF). The reagent is a secondary antibody with a toxic group MMAF, and when the primary antibody is combined and the toxic group is released in cells to kill target cells along with internalization of the antibody, the mechanism is similar to that of ADC. The specific method comprises the following steps: SK-BR-3 cells were collected, centrifuged, supernatant was discarded, cells were suspended in medium, and cell concentration was adjusted to 1X 105The volume is/mL. The cell suspension was added to a 96-well plate at 50. mu.l/well, and then incubated overnight in a 37 ℃ incubator. 4X test antibody was prepared at a maximum concentration of 40nM (4X), 5-fold dilutions, totaling 8 dilutions. A4 XMMAF solution (4. mu.g/mL) was prepared. To 96-well plates containing cells, respectively25. mu.l of 4 Xtest antibody and 25. mu.l of 4 XMMAF solution were added thereto and incubated at 37 ℃ for 72 hours in a constant temperature incubator. To the wells, 100. mu.l of CTG solution was added, and the luminescence signal of CTG was read with a microplate reader. The results in figure 17 show that PR003369 has the highest internalization-mediated cytotoxic activity compared to the control antibody RP000014 and other antibodies.
Example 6 Antibody Drug Conjugation (ADC)
This example prepared ADCs using ADC coupling technology cross-linking a toxic group MMAE to an anti-B7H4 antibody (PR003369 antibody or control antibody 1). The purity parameters of the product are as follows, and the HPLC detection method is the same as 1.7.2.
TABLE 6-1 ADC preparation of H2L2 antibody against B7H4
Figure PCTCN2021102952-APPB-000014
To investigate whether the binding activity of the antibody to the B7H4 target was affected after cross-linking of the MMAE groups, an antibody binding experiment was performed at the cellular level using the cell line MDA-MB-468, which highly expresses human B7H4, as in example 2. The results in fig. 18 show that MMAE group cross-linking does not substantially affect the B7H4 binding activity of the antibody on the tumor cell surface.
To investigate whether the antibody affected its internalization activity after cross-linking of MMAE groups, the internalization of the antibody was examined using the Zenon pHrodo iFL IgG labelling Reagents kit (Invitrogen, # Z25611). The experimental procedure was as in example 5. The results in fig. 19 show that MMAE group cross-linking does not substantially affect the internalizing activity of the antibody on tumor cells.
This example is to investigate the cell killing activity of antibody MMAE groups cross-linking ADCs. The cell line MDA-MB-468 highly expressing B7H4 was used as a target cell, and the killing efficiency was reflected by the detection of the conductivity of the target cell using an RTCA instrument from ACEA. The 96 well plate e-plate was first equilibrated with 50. mu.l of complete medium. MDA-MB-468 cells were digested, resuspended in RPM1640 complete medium containing 10% fetal bovine serum, and diluted to 1X 105mL, 50. mu.l/well in e-plate 96 plates, i.e.5X 103Perwell, incubated overnight at 37 ℃. The next day 100. mu.l of 2 Xconcentration-gradient diluted antibody was added to each well, with a maximum final concentration of 50nM and 8 concentrations of each antibody, in duplicate. The conductivity of the target cells was measured in real time and the target cell killing efficiency was calculated as (1-sample/blank) × 100% taking data from the 96 hour time point. The results in FIG. 20 show that the affinity matured variant PR003369-ADC has a higher tumor cell killing efficiency than the control antibody 1-ADC.
Example 7 determination of affinity of anti-B7H4 antibodies to human B7H4 recombinant protein
7.1. Determination of affinity by SPR method
10 XHBS-EP + (GE Healthcare, # BR-1006-69) was diluted 10-fold and used as an experimental buffer. The flow rate was set at 10. mu.l/min, and protein A was coupled to 4 channels of a chip CM5(GE Healthcare, # BR-1005-30): 1) setting the injection time to 800s, and injecting the mixture of 50mM NHS and 200mM EDC into 4 channels after fresh mixing at a volume ratio of 1: 1; 2) protein A was diluted to 20. mu.g/mL with sodium acetate (GE Healthcare, # BR-1003-50) at pH4.5 and injected into each channel for 800 s; 3) 1M ethanolamine 800s pH8.5 was injected to block the remaining active carboxyl groups on the chip surface. After blocking, the instrument was equilibrated for two additional hours with 1 XHBS-EP + buffer, and the final amount of protein A coupled was about 2000 RU.
A multi-cycle kinetic pattern was set up at Biacore T200, each cycle including capture of antibodies, binding of analytes, and regeneration of the chip. Antibodies PR002418, PR002421, control antibody 1, and control antibody 2 were all diluted to 1. mu.g/mL, injected into 2, 3, 4 channels at a flow rate of 10. mu.l/min for 30s, and each antibody was captured by a pre-conjugated ProteinA in an amount of about 160 RU. Human B7-H4(Sino biological, #10738-H08H) was injected into the four channels sequentially with a concentration gradient of 0nM, 1.5625nM, 3.125nM, 6.25nM, 12.5nM, 25nM, 50nM (one maximum concentration of 100nM was increased for control antibody 1) and the flow rate was set at 30. mu.l/min. The dissociation time was set to 200s for PR002418, PR002421 and control antibody 2, 500s for control antibody 1, and 180s for each injection. Finally, 10mM glycine-hydrochloric acid pH1.5(GE Healthcare, # BR-1003-54) was injected at the same flow rate for 30s to regenerate the chip.
The experimental results were analyzed using Biacore T200 analysis software 2.0, with channel 1 subtracted as the reference channel and the analytical model selected as a 1: 1 kinetic fitting model. The results are shown in Table 7-1 and A-D of FIG. 21, which shows that PR002421 has the highest protein affinity.
TABLE 7-1 affinity of anti-B7H4 antibodies for binding to human B7H4 protein (SPR method)
Antibodies Antigens Antibody concentration (nM) ka(1/Ms) kd(1/s) KD(M)
PR002418 Human B7H4(his tag) 50-1.5625 2.01E+05 4.75E-03 2.37E-08
PR002421 Human B7H4(his tag) 50-1.5625 3.50E+05 3.22E-04 9.21E-10
Control antibody
2 Human B7H4(his tag) 50-1.5625 2.61E+05 1.16E-03 4.45E-09
Control antibody
1 Human B7H4(his tag) 100-3.125 4.04E+04 1.89E-04 4.67E-09
7.2. Determination of affinity by BLI method
10 × kinetic buffer (ForteBio, #18-1105) was diluted to 1 × for affinity testing and dilution of antigen, antibody. Analysis of binding kinetics between antigen and antibody was performed by the biofilm interference (BLI) technique using an Octet Red96e (fortebio) molecular interaction analyzer.
When the affinity of the antigen and the antibody is measured, the sensor is rotated at 1000 rpm. The AHC sensors (Fortebio, #18-5060) placed in a column were equilibrated for 10 minutes in the test buffer before the AHC sensors were used to capture the B7-H4 antibodies at a capture height of 0.7 nm; AHC sensors in buffer after 120s of equilibrium and 2 times gradient dilution of human B7-H4 (concentration 50-3.125nM and 0nM) combined with 180s, dissociation 300 s. The AHC sensor was finally regenerated by immersion in 10mM glycine-hydrochloric acid pH1.5 solution to elute the proteins bound to the sensor.
When data Analysis is performed by using OctetData Analysis software (Fortebio, version 11.0), 0nM is used as a reference hole, a reference signal (reference subtraction) is subtracted, a 1: 1 Global fitting method is selected for data fitting, kinetic parameters of antigen-antigen binding protein binding are calculated, and k is obtainedon(1/Ms) value, kdis(1/s) value and KD(M) values (see Table 7-2), the results show a clear increase in protein affinity for the affinity matured variant PR003369 compared to PR 002418.
TABLE 7-2 affinity of anti-B7H4 antibodies for binding to human B7H4 protein (BLI method)
Antibodies Concentration (nM) KD(M) kon(1/Ms) kdis(1/s) FullR^2
PR002418 50-12.5 1.03E-08 2.75E+05 2.84E-03 0.9926
PR003369 50-12.5 1.39E-09 2.83E+05 3.94E-04 0.9981
Example 8 determination of epitope Competition for anti-B7H4 antibody binding to B7H4 Using the BLI method
Epitope competition experiments were performed on B7-H4 antibodies PR002418, PR002421, control antibody 1, and control antibody 2 using ForteBio Octet Red96e platform, and the experimental buffer was the same as described above. First, 100% signal of antibody is obtained: B7-H4(Acro Biosystems, # B74-H82E 2-200. mu.g) was captured at a capture height of 0.25nm using an SA sensor (Fortebio, # 18-5019). The sensor was equilibrated in buffer for 120s and then immersed in each antibody diluted to 100nM for 240s, and the final signal of antibody binding to B7-H4 was recorded as the 100% signal of the antibody. Step two, epitope competition experiment: B7-H4 was captured with an SA sensor at a capture height of 0.25 nm. The sensor was immersed in the primary antibody (at a concentration of 100nM) for a period of 240s, and then the SA sensor was immersed in a mixture of the primary and secondary antibodies (both antibodies at a final concentration of 100nM) for a period of 240s, and the difference in signal after immersion of the sensor in the antibody mixture was recorded as the signal of the antibody as the secondary antibody. The inhibition rate was calculated by the following formula,
inhibition ratio (%) - (A-B)/A100
A: 100% signal of an antibody (obtained from the first step), B: this antibody serves as the signal for the second antibody (obtained from the second step).
If the obtained inhibition ratio is greater than 85 (%), it means that the epitopes of the two antibodies are completely overlapped; if the inhibition ratio is less than 85 (%), it means that the epitopes bound by the two antibodies do not completely overlap.
The results in Table 8-1 show that PR002418 and PR002421 have different binding epitopes from B7-H4, and both have different epitopes from control antibody 1 and control antibody 2. Wherein PR002418 binds to one unique epitope (first epitope), PR002421 binds to another epitope (second epitope), and control antibody 1, control antibody 2 bind to the same epitope (third epitope).
TABLE 8-1 competitive assay for antibody binding to B7H4
Figure PCTCN2021102952-APPB-000015
Example 9 Cross-reactivity with other members of the B7 family
The B7 family proteins (see Table 9-1 for details) were diluted to 1. mu.g/mL with PBS, added to a 96-well plate (Corning, #9018) and incubated at 4 ℃ overnight at 100. mu.l per well. After discarding the liquid, the plate was washed 3 times with PBST buffer (pH7.4, containing 0.05% tween-20), 250. mu.l of 2% BSA blocking solution was added, and incubated at 37 ℃ for 1 hour. The blocking solution was discarded, and the plate was washed 3 times with PBST buffer (pH7.4, containing 0.05% Tween-20), and the antigen-binding protein to be tested was diluted to 2 concentrations of 10nM and 1nM, and 100. mu.l of each well was added and incubated at 37 ℃ for 1 hour with the same type of antibody as a control. PBST buffer (pH7.4, containing 0.05% Tween-20) after 3 times of washing, added with 5000 times diluted goat anti-human F (ab')2HRP secondary antibody (Jackson ImmunoResearch, 109-. After PBST buffer (pH7.4, containing 0.05% tween-20) was washed 3 times, 100. mu.l/well of TMB (Biopanda, # TMB-S-003) was added and left at room temperature in the dark for about 30 minutes; the reaction was stopped by adding 50. mu.l/well of a stop solution (BBI life sciences, # E661006-0200) to each well, and absorbance (OD450) at 450nm was measured in a microplate reader (PerkinElemer, # Enspire). FIG. 22 illustrates that the antibodies of the invention do not cross-react with other member proteins of the B7 family.
TABLE 9-1 Material information of B7 family protein used in this example
Other members of the B7 family Suppliers of goods Directory number
Human B7-1/CD80 Protein，Fc Tag(HPLC-verified) Acro B71-H5259
Human B7-2/CD86 Protein，Fc Tag Acro CD6-H5257
Human B7-DC/PD-L2/CD273 Protein，Recombinant(Fc Tag) Sino Biological H10292-H02H
Human PD-L1/B7-H1 Protein，His Tag(HPLC verified) Acro PD1-H5229
CD275/ICOS ligand Protein，Human，Recombinant(Fc Tag) Sino Biological 11559-H02H
B7-H3/CD276 Protein，Human，Recombinant(ECD，Fc Tag) Sino Biological 11188-H02H
Recombinant human B7H4 Fc Chimera R&D 8870-B7-050
Recombinant human VISTA/B7H5/PD-1H Fc Chimera R&D 7126-B7-050
B7-H6/NCR3LG1 Protein，Human，Recombinant(Fc Tag) Sino Biological 16140-H02H
Recombinant human B7H7/HHLA2 Fc Chimera R&D 8084-B7-050
Example 10 serum stability assay
Diluting 30 μ l antibody to 270 μ l normal human serum (serum concentration 90%), dividing antibody into 5 parts, respectively incubating at 37 deg.C for 0, 1, 4, 7, and 14 days, taking out, quick freezing with liquid nitrogen, and storing at-80 deg.C. The binding of the antibody to B7H4 on SK-BR-3 cells was detected by flow methods.
SK-BR-3 or CHOK1/hB7H4 cells were digested and resuspended in PBS containing 2% BSA. The cell density was adjusted to 1X 106cells/mL. Cells were seeded at 100. mu.L/well in 96-well V-plates (Corning, #3894), followed by the addition of 100. mu.L/well of test antibody diluted in a 3-fold concentration gradient 2-fold the final concentration. The cells were incubated at 4 ℃ for 2 hours in the dark. Thereafter, cells were rinsed twice with 100. mu.L/well of pre-chilled 2% BSA in PBS, centrifuged at 500g for 5 minutes at 4 ℃ and the supernatant discarded. mu.L/well fluorescent secondary antibody (Alexa Fluor488-conjugated affinity Goat Anti-Human IgG, Fc gamma Fragment specificity, Jackson, # 109-. Cells were washed twice with 100. mu.L/well pre-chilled 2% BSA in PBS, centrifuged at 500g for 5 min at 4 ℃ and the supernatant discarded. Finally, cells were resuspended in 200. mu.L/well pre-cooled 2% BSA in PBS and the fluorescence emission signal was read using an ACEA Novocyte3000 flow cytometer. The results in FIG. 23 show that anti-B7H4 antibodies PR002418, PR002037, and PR003369, as well as control antibody 1 and control antibody 2, have good stability in human serum at 37 ℃ for 14 days.
Example 11 pharmacokinetics in C57BL/6 mice
Selecting 6 female Nu/Nu mice with the weight of 18-22 g, and injecting the drugs through tail vein according to the dose of 20 mg/kg; one group 3 collected whole blood before and 15 minutes, 24 hours (1 day), 4 days, and 10 days after the administration, and the other group 3 collected whole blood only before and 5 hours, 2 days, 7 days, and 14 days after the administration. The whole blood was allowed to stand for 30 minutes to allow it to clot, then centrifuged at 2,000rpm for 5 minutes at 4 ℃ and the separated serum sample frozen at-80 ℃ until analysis. This example uses an ELISA method to quantitatively determine drug concentrations in mouse serum. The ELISA Fc end overall detection method is characterized in that a goat anti-human Fc polyclonal antibody coated on a 96-well plate is used for capturing fusion protein containing human Fc in mouse serum, and then a goat anti-human Fc secondary antibody marked by HRP is added for detection. The plasma concentration data were analyzed using the Phoenix WinNonlin software version 8.2, using a non-compartmental model (NCA) to evaluate pharmacokinetics.
Table 11-1 is the pharmacokinetic parameters of PR002418, PR002421 and control antibody 2(PR 002962). The results in fig. 24 show that the half-life of PR002418 in mice is about 6.56 days, PR002421 in mice is about 6.64 days, and control antibody 2 in mice is about 5.90 days, as calculated from the data of the first 14 days under the Fc-terminal global assay method. This result indicates that PR002418 and PR002421 have slightly longer half-lives in mice than control antibody 2.
TABLE 11-1 pharmacokinetic parameters of anti-B7H4 antibodies
PK parameters PR002421 PR002418 PR002962
T1/2(hr) 159.3 157.5 141.5
Vd(mL/kg) 93.3 89.3 93.3
AUCall(μg/mL hr) 36，710 37，786 33，913
C1(mL/hr/kg) 0.42 0.41 0.48
C0(μg/mL) 437.9 387.6 440.4
Example 12 anti-tumor efficacy of anti-B7H4 antibodies
BALB/c nude mouse MDA-MB-468 tumor model
Cell inoculation day Each BALB/c nude mouse was inoculated subcutaneously with 1X 10 cells7MDA-MB-468 tumor cells, which were resuspended in PBS/Matrigel (1: 1) mixture (0.1 mL/cell), were inoculated subcutaneously. When the average tumor volume of the mice is 135mm3The groups were divided into 5 groups of 25 mice, and the administration period was 2 times per week, and 12 times of administration were performed in total, i.e., intraperitoneal administration was performed. After the start of the administration, body weight and tumor volume were weighed twice a week in the following manner: tumor volume (mm)3) 0.5 × tumor major diameter × tumor minor diameter2. The experiment was terminated on day 39 post-dose and all mice were euthanized.
BALB/c nude mouse MDA-In vivo anti-tumor efficacy of the MB-468 tumor model referring to FIG. 25, in particular, the 39 th balance mean tumor volume of the vehicle control group mice after administration was 1054mm3. Test agent PR002418(5mg/kg) treatment group the 39 th balance mean tumor volume after administration was 606mm3Compared with the vehicle control group, the tumor inhibition rate TGI (%) is 42.45 percent, and the difference is significant (the p value is 0.015). Test agent PR002418(15mg/kg) treatment group the 39 th balance mean tumor volume after administration was 532mm3Compared with the vehicle control group, the tumor inhibition rate TGI (%) is 49.47% with a significant difference (p value is 0.007). Test drug PR002421(5mg/kg) treatment group mean tumor volume 665mm at 39 th day after dosing3Compared with the vehicle control group, the tumor inhibition ratio TGI (%) is 36.86%, and the difference is not significant (p value is 0.07). Test agent PR002421(15mg/kg) the treatment group had a mean tumor volume of 335mm after administration at 39 th day3Compared with the vehicle control group, the tumor inhibition rate TGI (%) was 68.23%, which was significantly different (p value was 0.018).
NSG mice reconstitute MDA-MB-468 tumor model of human PBMC immune system
Cell inoculation day Each NCG mouse was inoculated subcutaneously 5X 106MDA-MB-468 tumor cells, which were resuspended in PBS/Matrigel (1: 1) mixture (0.1 mL/cell), were inoculated subcutaneously. When the average tumor volume of the mice is 126mm3Grouping was performed, and 30 mice were divided into 5 groups, each mouse was inoculated intravenously at 5X 106Human PBMC, cells were resuspended in 200. mu.l PBS. The following day, the administration period was twice a week, 8 administrations were performed, and the abdominal cavity administration was performed. After the start of the administration, body weight and tumor volume were weighed twice a week in the following manner: tumor volume (mm)3) 0.5 × tumor major diameter × tumor minor diameter2. Experimental observations were terminated at day 36 post-dose, and all mice were subsequently euthanized.
In vivo anti-tumor effect of NSG mice in the MDA-MB-468 tumor model for reconstitution of human PBMC immune system is shown in FIG. 26, in particular, the 36 th balance mean tumor volume of the vehicle control mice after administration is 942mm3. Test agent PR002418(15mg/kg) treatment group after administrationThe 36 th balance mean tumor volume was 585mm3Compared with the vehicle control group, the tumor inhibition ratio TGI (%) is 37.91%, and the difference is not significant (p value is 0.073). Test agent PR002421(15mg/kg) the treatment group had a 36 th day balance mean tumor volume of 670mm after administration3Compared with the vehicle control group, the tumor inhibition ratio TGI (%) is 28.87% without significant difference (p value is 0.200). Test agent PR003369(15mg/kg) treatment group 36 th day after administration has a mean tumor volume of 354mm3Compared with the vehicle control group, the tumor inhibition ratio TGI (%) is 62.38 percent, and the difference is significant (the p value is 0.008). Test drug control antibody 2(15mg/kg) treatment group had a 36 th day mean tumor volume of 533mm after dosing3Compared with the vehicle control group, the tumor inhibition ratio TGI (%) was 43.41% with a significant difference (p value of 0.028).
Example 13 immunohistochemical staining (IHC)
The pathological tissue chip was purchased from Guilin Pan-spectral Biotechnology, Inc. Comprises a BRC1021 breast cancer tissue chip, an EMC1021 endometrial cancer tissue chip, an OVC1021 ovarian cancer tissue chip and an MNO1021 normal tissue chip. Paraffin sections were 4um thick and were taken with positive control tissue. Dewaxing and washing; antigen retrieval: PH6 (citric acid), heated at 125 deg.C for 5 min, sealed for 10 min, and cooled at room temperature for 30 min; after washing with water, 0.3% hydrogen peroxide for 5 minutes, and then washing with TBST for 3 times for 5 minutes; dako blocking solution was used directly, and blocked with an incubation chamber at room temperature for 20 min; removing the confining liquid, adding the primary antibody, directly using the antibody diluent Dako, incubating for 60 minutes in an incubation box at room temperature, and replacing a control with Rabbit IgG; TBST three times, five minutes each time; a secondary antibody, Anti-Rabbit (EnVision + System-HRP Labelled Polymer), incubated with the incubation cassette for 30 minutes at room temperature; TBST three times, five minutes each time; DAB coloration, 0.85mL of distilled water, adding 50 mul of reagents according to the sequence of the reagents ABC, and incubating for 5 minutes by using an incubation box at room temperature; washed with distilled water and counterstained with hematoxylin. Observing under a microscope, sealing and reading.
The results in FIG. 27 show that B7H4 was slightly expressed in adrenal gland, renal cortex, bladder, mammary gland, fallopian tube, esophagus, ureter and endometrium of normal tissues, and not expressed in other tissues (FIG. 27, A). In contrast, it was highly expressed on breast cancer, ovarian cancer and endometrioma (fig. 27, B). For example, in ovarian cancer, IHC scores 67.65% of 102 total samples on a scale of 2-4, and in endometrioma, IHC scores 62.24% of 98 total samples on a scale of 2-4 (fig. 27, C).
Example 14 Structure and design of B7H4 × CD3 bispecific antibody
Selected anti-B7H4 antibodies and anti-CD 3 antibodies were used to make bispecific antibodies. The preparation of this B7H4 XCD 3 bispecific antibody the diabody can simultaneously bind to two targets, wherein one end can recognize B7H4 specifically expressed on the surface of tumor cells, and the other end can bind to a CD3 molecule on T cells. When the B7H4 XCD 3 double antibody binds to the surface of the tumor cells, T cells near the tumor cells can be recruited and activated, thereby killing the tumor cells.
FIGS. 28A and B are "1 + 1" Fab-Fc-scFv asymmetric structural molecules; c and D are B7H4 x CD3 double antibody molecules with '1 + 1' Fab-Fc-crossFab asymmetric structure. For the "1 + 1" asymmetric structure molecule, structures (1) and (2) involve three protein chains, which comprise the heavy and light chains of the corresponding anti-B7H4 antibody, respectively, and the scFv polypeptide chain of the anti-CD 3 antibody described above (see fig. 28, a and B). For the "1 + 1" asymmetrically-structured molecule, structures (3) and (4) involve four protein chains, which comprise the heavy and light chains of the corresponding anti-B7H4 antibody, respectively, and the heavy and light chains of the anti-CD 3 antibody described above (see FIGS. 28, C and D).
E and F, G and H, I and J are B7H4 × CD3 double antibody molecules with "2 + 1" asymmetric structure. For the "2 + 1" asymmetrically-structured molecule, structures (5) and (6) involve four protein chains, which comprise the heavy and light chains of the corresponding anti-B7H4 antibody, respectively, and the heavy and light chains of the anti-CD 3 antibody described above (see FIGS. 28, E and F). For the "2 + 1" asymmetric structure molecule, structures (7) (8) and (9) (10) involve three protein chains comprising the heavy and light chains of the corresponding anti-B7H4 antibody, respectively, and the polypeptide chains of the scFv of the above-described anti-B7H4 and CD3 antibodies (see fig. 28, G, H, I and J).
To minimize the byproduct formation of heavy chains with mismatches (e.g., two heavy chain mismatches of an anti-CD 3 antibody), a mutated heterodimeric Fc region is used, carrying a "knob-hole" mutation and engineered disulfide bonds, as described in WO2009080251 and WO 2009080252. The B7H4 × CD3 bispecific antibody has an Fc of IgG1 and carries L234A, L235A and P329G (numbered according to the EU index) mutations on CH3 of the Fc. Each bispecific antibody was generated by co-transfecting three or four different mammalian expression vectors simultaneously, encoding: 1) the heavy chain of the corresponding anti-B7H4 antibody, which carries a "Hole" mutation in the Fc region to produce heterodimeric antibodies, Fc CH3 carries L234A, L235A and P329G mutations. 2) The heavy chain of the corresponding anti-CD 3 antibody, which carries the "knob" mutation in the Fc region to produce heterodimeric antibodies, the CH3 of Fc carries the L234A, L235A, P329G mutations. 3) The light chain of the corresponding anti-CD 3 antibody. 4) The light chain of the corresponding anti-B7H4 antibody. The "knob" mutation in the human IgG1Fc region consisted of: T366W, the "Hole" mutation consists of: T366S, L368A, Y407V. In addition, S354C and "Hole" Y349C, which may include a "knob" Fc region, form a pair of disulfide bonds to increase stability and heterodimeric antibody production.
Table 14-1, Table 14-2, and Table 14-3 set forth the B7H4 XCD 3 diabodies constructed in this example, with the structure numbers in the tables corresponding to FIG. 28. Tables 14-4 are the sequences of the linker peptides. The sequence numbers corresponding to the CD3 monoclonal antibody molecules are listed in tables 14-5. The B7H4 monoclonal antibody molecule is derived from tables 1-7. Table 14-6 lists the sequence numbers corresponding to the B7H4 × CD3 diabodies. Tables 14-7 list the sequence numbers of the corresponding CDR sequences of the first and second antigen-binding domains of the diabodies.
TABLE 14-1B 7H4 XCD 3 diabodies with "1 + 1" Fab-Fc-scFv asymmetric structure
Figure PCTCN2021102952-APPB-000016
TABLE 14-2B 7H4 XCD 3 diabodies with "1 + 1" Fab-Fc-crossFab asymmetric structure
Figure PCTCN2021102952-APPB-000017
Figure PCTCN2021102952-APPB-000018
TABLE 14-3B 7H4 XCD 3 diabodies with "2 + 1" asymmetric structure
Figure PCTCN2021102952-APPB-000019
TABLE 14-4 linker peptide sequences
Linker peptide names Length of Sequence of
GS_5 5 GGGGS SEQ ID NO：133
GS_15 15 GGGQSGQGGSGGGGS SEQ ID NO：134
GS_20 20 GGGGSGGGGSGGGGSGGGGS SEQ ID NO：135
TABLE 14-5 sequence numbering of the CD3 antibody of this example
Antibody numbering Light chains Heavy chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
PR000627 131 86 76 46 53 63 8 20 34
PR001924 111 97 86 78 46 53 63 10 20 34
PR001848 110 96 86 78 46 53 63 10 20 34
PR003886 110 107 86 84 46 53 63 10 20 34
TABLE 14-6 sequence numbering of the B7H4 XCD 3 diabodies of this example
Antibody numbering Polypeptide chain-1 Polypeptide chain-2 Polypeptide chain-3 Polypeptide chain-4
PR002849 109 118 119
PR002850 114 120 119
PR002851 114 121 119
PR002852 115 122 119
PR002855 97 123 118 109
PR002856 97 123 120 114
PR002857 97 123 121 114
PR002858 97 123 122 115
PR002987 97 124 118 109
PR002994 97 125 118 109
PR003001 126 118 109
PR003008 127 118 109
PR003733 110 128 129
PR003899 110 130 129
TABLE 14-7B 7H4 XCD 3 Dual antibody molecules CDR sequence numbering table
Figure PCTCN2021102952-APPB-000020
Figure PCTCN2021102952-APPB-000021
TABLE 14-8B 7H4 XCD 3 Dual-antibody protein expression
Figure PCTCN2021102952-APPB-000022
Example 15 FACS detection of the ability of B7H4 × CD3 diabodies to bind to B7H4
SK-BR-3 cells were digested. T cells were isolated using the human T cell isolation kit (Miltenyi, #130-096-535) as described in the specification. Resuspended in PBS containing 2% BSA. The cell density was adjusted to 1X 106cells/mL. Cells were seeded at 100. mu.L/well in 96-well V-plates (Corning, #3894), followed by the addition of 100. mu.L/well of test antibody diluted in a 3-fold concentration gradient 2-fold the final concentration. Cells were incubated at 4 ℃ for 2 hours in the dark. Thereafter, cells were rinsed twice with 100. mu.L/well of pre-chilled 2% BSA in PBS, centrifuged at 500g for 5 minutes at 4 ℃ and the supernatant discarded. Then 100. mu.L/well fluorescent secondary antibody (Alexa Fluor488-conjugated affinity assay Goat Anti-Human IgG, Fc gamma Fragment specificity, Jackson, # 109-. Cells were washed twice with 100. mu.L/well pre-chilled 2% BSA in PBS, centrifuged at 500g for 5 min at 4 ℃ and the supernatant discarded. Finally, cells were resuspended in 200. mu.L/well pre-cooled 2% BSA in PBS and the fluorescence emission signal was read using an ACEA Novocyte3000 flow cytometer.
The results of the "1 + 1" asymmetric structure of B7H4 × CD3 diabody binding to SK-BR-3 cells are shown in FIG. 29, A-C. The result shows that the double-antibody molecule has better binding activity with B7H4 on the surface of the tumor cell SK-BR-3. FIG. 29A shows the binding activity of the double antibodies against ScFv polypeptide chain at the end of B7H4 and against CD3, wherein the double antibodies PR002849, PR002850 and PR002851 against ScFv polypeptide chain of PR001476 and PTM mutant antibody at the end of B7H4 have stronger binding activity than the double antibodies PR002852 against ScFv polypeptide chain of PR002037 PTM mutant antibody at the end of B7H 4; FIG. 29B shows the binding activity of the diabody molecules with Fab structure at the anti-B7H4 end and Cross Fab structure at the anti-CD 3 end, wherein the diabody molecules PR002855, PR002856 and PR002857 derived from PR001476 and PTM mutant antibody at the anti-B7H4 end have stronger binding activity than the diabody molecule PR002858 derived from PR002037 PTM mutant antibody at the anti-B7H4 end; fig. 29C shows the binding activity of a double antibody molecule in which the anti-B7H4 end is an ScFv polypeptide chain and the anti-CD 3 end is an Fab structure, and PR003733 and PR003899 are the same ScFv polypeptide chains at the anti-B7H4 end, and the binding activities are the same. The results of the "2 + 1" asymmetric structure of B7H4 × CD3 diabody binding to SK-BR-3 cells are shown in FIG. 29, D. The result shows that the 2-valent anti-B7H4 end also has high binding activity. The results of the "1 + 1" asymmetric structure of B7H4 × CD3 diabody binding human T cells are shown in FIG. 29, E-G; the results of the "2 + 1" asymmetric structure of the B7H4 xcd 3 diabody binding human T cells are shown in fig. 29, H. The results show that the anti-CD 3 terminal is Fab structure or ScFv polypeptide chain can be combined on the surface of T cell, while the weak anti-CD 3 terminal double-antibody PR003899 can not be combined at FACS level.
Example 16T cell killing experiment
In the experiment, human primary T cells are used as effector cells, and a cell line SK-BR-3 with high expression of B7H4, or a cell line HCC-1954 expressed in MDA-MB-468 and B7H4, or a cell line MDA-MB-231 with negative expression of B7H4 are used as target cells. The killing efficiency was reflected by the detection of the conductivity of the target cells using the ACEA RTCA instrument. The 96-well plate e-plate was first equilibrated with 50. mu.l of complete medium. Target cells were digested, resuspended in RPM1640 complete medium containing 10% fetal bovine serum, and diluted to 4X 105mL, 50. mu.l/well in e-plate 96 plates, i.e.2X 104Incubate overnight at 37 ℃ per well. The next day primary T cells were isolated using the MeitianNi T cell isolation kit (Miltenyi, #130-096-535) according to the protocol. Fresh 50. mu.l of 2X 10 solution per well5T cell culture medium, then 50 u l4 x concentration gradient dilution of antibody, antibody maximum final concentration of 10nM, each antibody total 8 concentrations, set up two repeat. The conductivity of the target cells was measured in real time and the target cell killing efficiency was calculated as (1-sample/blank) × 100% typically from the data at the 24 hour time point. The supernatant was collected for 24 hours,ELISA method for detecting IFN-gamma concentration. The ELISA detection method was performed according to the instructions of the IFN gamma Human Uncoated ELISA Kit (Thermo, # 88-7316-77).
The results of the B7H4 XCD 3 diabody with the asymmetric structure of "1 + 1" activating T cells and killing target cells SK-BR-3 are shown in FIG. 30, A-K. A, B in FIG. 30 shows that the anti-B7H4 terminal is Fab structure, the anti-CD 3 terminal is the killing activity of the double anti-molecule of ScFv polypeptide chain and the generation of cytokine IFN-gamma, wherein the anti-B7H4 terminal is derived from PR002037 PTM mutant antibody, and the double anti-molecule PR002852 of ScFv polypeptide chain has the strongest killing activity and the generation of cytokine IFN-gamma. C, D in FIG. 30 shows the killing activity and cytokine IFN-. gamma.production of a dual-antibody molecule with Fab structure at the anti-B7H4 end and Cross Fab structure at the anti-CD 3 end, wherein PR002852, a dual-antibody molecule derived from PR002037 PTM mutant antibody at the anti-B7H4 end, has the strongest killing activity and cytokine IFN-. gamma.production. E, F in FIG. 30 shows that the anti-B7H4 end is ScFv polypeptide chain, the anti-CD 3 end is Fab structure, the double-antibody molecule killing activity and cytokine IFN-gamma generation are higher than those of the double-antibody molecule PR003733 with strong CD3 end and the double-antibody molecule PR003899 with weak CD3 end. FIG. 30G-L compares the killing activity and cytokine secretion of PR003733 and PR003899 on other tumor cells MDA-MB-468, HCC-1954 and MDA-MB-231, and shows that PR003733 is higher than PR003899 in MDA-MB-468, HCC-1954 cell killing in vitro and cytokine IFN-gamma production; MDA-MB-231 was a negative control cell that did not express B7H4, and neither antibody had any effect. The results of the "2 + 1" asymmetric structure of B7H4 × CD3 diabody activated T cells and killed the target cell SK-BR-3 are shown in FIG. 30, M-N. The double-antibody molecules with the asymmetric structure of 2+1 can kill SK-BR-3 cells and generate cytokine IFN-gamma, and the double-antibody molecules PR003001 and PR003008 with the Cross Fab structure at the anti-CD 3 end have stronger killing activity and cytokine IFN-gamma generation than the double-antibody molecules PR002987 and PR002994 with ScFv polypeptide chain at the anti-CD 3 end.
Example 17 NSG mice reconstitute tumor models of the human PBMC immune System
Cell inoculation day Each NCG mouse was inoculated subcutaneously 5X 106MDA-MB-468 tumor cells, which were resuspended in PBS/Matrigel (1: 1) mixture (0.1 mL/cell), were inoculated subcutaneously. When the average tumor volume of the mice is 126mm3Grouping was performed, 18 mice were divided into 3 groups, and each mouse was inoculated intravenously at 5X 106Human PBMC, cells were resuspended in 200. mu.l PBS. The following day was started with a weekly administration cycle of 3 total administrations, i.v. administration. After the start of the administration, body weight and tumor volume were weighed twice a week in the following manner: tumor volume (mm)3) 0.5 × tumor major diameter × tumor minor diameter2. Experimental observations were terminated at day 36 post-dose, followed by euthanasia treatment of all mice.
The 36 th balance mean tumor volume of the vehicle control group mice after administration is 942mm3. Test agent PR003733(2mg/kg) treatment group, 36 th balance mean tumor volume 590mm after dosing3Compared with the vehicle control group, the tumor inhibition ratio TGI (%) is 37.31%, with a significant difference (p value of 0.048). Test agent PR003899(2mg/kg) treatment group had a 36 th balance mean tumor volume of 0mm after administration3The tumors regressed completely, with a significant difference (p value of 0.0001) compared to the vehicle control group, and the tumor inhibition TGI (%) was 100% (see fig. 31, a).
In the HCC-1954 model, each NCG mouse was inoculated subcutaneously 5X 10 on the day of cell inoculation6HCC-1954 tumor cells, resuspended in PBS/Matrigel (1: 1) mixture (0.1 mL/cell), and inoculated subcutaneously. When the average tumor volume of the mice is 102mm3Grouping was performed, 15 mice were divided into 3 groups, and each mouse was inoculated intravenously at 3X 106Human PBMC, cells were resuspended in 200. mu.l PBS. The following day, once a week, 2 doses were administered, intravenously. After the start of the administration, body weight and tumor volume were weighed twice a week in the following manner: tumor volume (mm)3) 0.5 × tumor major diameter × tumor minor diameter2. The experimental observations were terminated on day 16 post-dose, and all mice were subsequently euthanized.
The 16 th balance mean tumor volume of the vehicle control group mice after administration is 622mm3. Test drug PR003733(0.5mg/kg) treatment group 16 th day balance mean tumor volume 450mm after dosing3Compared with the vehicle control group, the tumor inhibition ratio TGI (%) is 27.64% without significant difference (p value is 0.1). Test drug PR003899(0.5mg/kg) treated group 16 th day balance mean tumor volume 322mm after dosing3Compared with the vehicle control group, the tumor inhibition ratio TGI (%) was 48.27% (see fig. 31, B) with a significant difference (p value of 0.0028).
Pharmacodynamic experiments of the two tumor models show that the efficacy of PR003899 is better than that of PR 003733.

Claims (22)

  1. An antibody or variant thereof targeting B7H4, wherein said antibody comprises a light chain variable region and a heavy chain variable region, wherein:
    the light chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 47. LCDR1, LCDR2 and LCDR3 shown at 54 and 64; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 21 and 35; or the like, or, alternatively,
    the light chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 48. LCDR1, LCDR2 and LCDR3 shown at 54 and 65; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 11. HCDR1, HCDR2 and HCDR3 shown at 22 and 36;
    the variant has an amino acid mutation in the light chain variable region and/or the heavy chain variable region of the antibody and is capable of maintaining the function of the antibody; preferably, the amino acid mutation is an amino acid substitution, and the number of the amino acid substitutions is 1 to 3.
  2. The antibody or variant thereof of claim 1, wherein the variant is an amino acid substitution at position 3 or4 of HCDR2, position 3 of HCDR3, and/or an amino acid substitution at position 4 of LCDR3 in the heavy chain variable region of the antibody; preferably, the first and second electrodes are formed of a metal,
    the light chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 48. LCDR1, LCDR2 and LCDR3 shown at 55 and 66; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 11. HCDR1, HCDR2 and HCDR3 shown at 22 and 36; or the like, or, alternatively,
    the light chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 47. LCDR1, LCDR2 and LCDR3 shown at 54 and 67; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 23 and 35; or the like, or, alternatively,
    the light chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 47. LCDR1, LCDR2 and LCDR3 shown at 54 and 67; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 24 and 35; or the like, or, alternatively,
    the light chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 47. LCDR1, LCDR2 and LCDR3 shown at 54 and 69; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 9. 24 and 38, HCDR1, HCDR2, and HCDR 3.
  3. The antibody or variant thereof of claim 1 or 2, wherein the heavy chain variable region further comprises a heavy chain variable region framework region HFWR, and/or the light chain variable region further comprises a light chain variable region framework region LFWR, wherein the HFWR is the heavy chain variable region framework region of a human antibody and the LFWR is the light chain variable region framework region of a human antibody; preferably, the first and second electrodes are formed of a metal,
    the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 87; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 77; or the like, or, alternatively,
    the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 88; the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 79; or the like, or a combination thereof,
    the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 89; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79; or the like, or, alternatively,
    the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 90; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 80; or
    The light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 90; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 81; or
    The light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 93; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 83; or
    The light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 91; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79, or a pharmaceutically acceptable salt thereof.
  4. The antibody or variant thereof of any one of claims 1-3, wherein the antibody further comprises a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region of the antibody is selected from the group consisting of hIgG1, hIgG2, hIgG3 or hIgG4 and the light chain constant region is selected from the group consisting of a kappa chain or a lambda chain; more preferably, the variant has an amino acid substitution at position 239 and/or 332 of the Fc of the antibody, preferably the amino acid substitution is S239D and/or I332E.
  5. The antibody or variant thereof of any of claims 1-4, wherein the antibody is a full length antibody, Fab ', F (ab')2Fv, scFv, or a monoclonal antibody or a polyclonal antibody produced from the above antibody.
  6. The antibody of claim 5, which comprises
    (1) A heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 95; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 109; or, the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 98; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 112; or the like, or, alternatively,
    the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 98; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 113; or the like, or, alternatively,
    the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 99; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or, alternatively,
    the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 100; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or, alternatively,
    the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 101; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 109, or a pharmaceutically acceptable salt thereof; or the like, or, alternatively,
    the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 102; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or, alternatively,
    the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 106; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 117; or the like, or, alternatively,
    the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 98; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 115; or the like, or, alternatively,
    the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 103; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 112; or the like, or, alternatively,
    the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 103; the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 115; or the like, or, alternatively,
    (2) it comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 132.
  7. A bispecific antibody targeting B7H4, comprising a protein a functional region and a protein B functional region, wherein the protein a functional region is the antibody targeting B7H4 of any one of claims 1 to 6; the B protein functional region is an antibody which is not targeted to B7H 4; preferably, the non-B7H 4-targeting antibody is a CD 3-targeting antibody; more preferably, the CD 3-targeting antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises amino acid sequences represented by SEQ ID NOs: 46. LCDR1, LCDR2 and LCDR3 shown at 53 and 63; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; or, the light chain variable region comprises an amino acid sequence consisting of SEQ ID NOs: 46. LCDR1, LCDR2 and LCDR3 shown at 53 and 63; the heavy chain variable region comprises amino acid sequences consisting of SEQ ID NOs: 10. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; even more preferably, in the CD 3-targeting antibody, the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 86; the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; or, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 86; the VH of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78; or, the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 86; the VH of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 84.
  8. The bispecific antibody of claim 7, wherein the protein B functional region comprises a light chain variable region and a heavy chain variable region, and the protein A functional region comprises a light chain variable region and a heavy chain variable region; wherein the content of the first and second substances,
    in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 64, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 21 and 35; or the like, or, alternatively,
    in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, a light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 67, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 23 and 35; or the like, or, alternatively,
    in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 67, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 24 and 35; or the like, or, alternatively,
    in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 8. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 48. 54 and 65, LCDR1, LCDR2, and LCDR3, the heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 11. HCDR1, HCDR2 and HCDR3 shown at 22 and 36; or the like, or, alternatively,
    in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 64, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 21 and 35; or the like, or, alternatively,
    in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 67, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 23 and 35; or the like, or, alternatively,
    in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 47. 54 and 67, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9. HCDR1, HCDR2 and HCDR3 shown at 24 and 35; or the like, or, alternatively,
    in the functional region of the protein B, the variable region of the light chain comprises amino acid sequences respectively represented by SEQ ID NO: 46. 53 and 63, and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 10. HCDR1, HCDR2 and HCDR3 shown at 20 and 34; in the protein A functional region, the light chain variable region comprises amino acid sequences respectively represented by SEQ ID NO: 48. 54 and 65, LCDR1, LCDR2, and LCDR3, the heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 11. 22 and 36, HCDR1, HCDR2, and HCDR 3.
  9. The bispecific antibody of claim 8, wherein the protein B functional region comprises a light chain variable region and a heavy chain variable region, and the protein a functional region comprises a light chain variable region and a heavy chain variable region; wherein the content of the first and second substances,
    in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 87 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 77; or the like, or, alternatively,
    in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 80; or the like, or, alternatively,
    in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 81; or the like, or, alternatively,
    in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 76; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79; or the like, or, alternatively,
    in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 87 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 77; or the like, or, alternatively,
    in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 80; or the like, or, alternatively,
    in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 81; or the like, or, alternatively,
    in the functional region of the B protein, the variable region of the light chain comprises the amino acid sequence shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 78; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79; or the like, or, alternatively,
    in the B protein functional region, a light chain variable region comprises a light chain variable region shown as SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 84; in the protein A functional region, a light chain variable region comprises a sequence shown as SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 79, or a pharmaceutically acceptable salt thereof.
  10. The bispecific antibody of claim 9, wherein the bispecific antibody is selected from the group consisting of:
    (1) the bispecific antibody comprises three polypeptide chains: wherein the first polypeptide chain comprises a sequence as set forth in SEQ ID NO: 109; the second polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the like, or, alternatively,
    the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 114; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 120; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the like, or, alternatively,
    the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 114; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 119; or the like, or, alternatively,
    the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 115; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 122; the third polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 119; or the like, or, alternatively,
    the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 126, or a pharmaceutically acceptable salt thereof; the second polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 109, or a pharmaceutically acceptable salt thereof; or the like, or a combination thereof,
    the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 127; the second polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or the like, or, alternatively,
    the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 110; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 128; the third polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 129; or the like, or a combination thereof,
    the first polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 110; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 130; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 129;
    (2) the bispecific antibody comprises four polypeptide chains: wherein the first polypeptide chain comprises a sequence as set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the fourth polypeptide chain comprises the amino acid sequence set forth as SEQ ID NO: 109, or a pharmaceutically acceptable salt thereof; or the like, or a combination thereof,
    the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 120; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or, alternatively,
    the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 121; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 114; or the like, or, alternatively,
    the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 122; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 115; or the like, or a combination thereof,
    the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 124; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 109, or a pharmaceutically acceptable salt thereof; or the like, or, alternatively,
    the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the fourth polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 109, or a pharmaceutically acceptable salt thereof.
  11. A chimeric antigen receptor comprising the antibody of any one of claims 1 to 6 or the bispecific antibody of any one of claims 7 to 10.
  12. A genetically modified cell comprising the chimeric antigen receptor of claim 11; the cell is preferably a eukaryotic cell, more preferably an isolated human cell, even more preferably an immune cell such as a T cell or NK cell.
  13. An isolated nucleic acid encoding the antibody of any one of claims 1-6, the bispecific antibody of any one of claims 7-10, or the chimeric antigen receptor of claim 11.
  14. An expression vector comprising the isolated nucleic acid of claim 13.
  15. A transformant comprising the expression vector of claim 14; the transformant is prepared by transforming the recombinant expression vector into a host cell, wherein the host cell is preferably a prokaryotic cell or a eukaryotic cell.
  16. A method of making an antibody or bispecific antibody targeting B7H4, comprising the steps of: culturing the transformant as described in claim 15, and obtaining the antibody or bispecific antibody targeting B7H4 from the culture.
  17. An antibody drug conjugate comprising an antibody according to any one of claims 1 to 6, a bispecific antibody according to any one of claims 7 to 10 and a conjugate moiety including, but not limited to, a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, wherein the antibody moiety and the conjugate moiety are conjugated via a chemical bond or a linker.
  18. A pharmaceutical composition comprising the antibody of any one of claims 1 to 6 or the bispecific antibody of any one of claims 7 to 10 and optionally a pharmaceutically acceptable carrier.
  19. Use of an antibody according to any one of claims 1 to 6, a bispecific antibody according to any one of claims 7 to 10, a chimeric antigen receptor according to claim 11, an immune cell according to claim 12, an antibody drug conjugate according to claim 17 or a pharmaceutical composition according to claim 18 for the manufacture of a medicament, a kit and/or a dosing device for the treatment and/or prevention of cancer; preferably, the cancer is a B7H 4-positively expressed tumor; more preferably breast cancer, such as triple negative breast cancer, ovarian cancer and endometrioma.
  20. A method for detecting B7H4 in a sample, comprising detecting using the antibody of any one of claims 1 to 6, the bispecific antibody of any one of claims 7 to 10; preferably, the detection method is for non-diagnostic purposes.
  21. A kit comprising the antibody of any one of claims 1 to 6, the bispecific antibody of any one of claims 7 to 10, the chimeric antigen receptor of claim 11, the immune cell of claim 12, the antibody drug conjugate of claim 17 and/or the pharmaceutical composition of claim 18, and optionally, instructions.
  22. A drug delivery device, said drug delivery device comprising: (1) an infusion module for administering the pharmaceutical composition of claim 18 to a subject in need thereof, and (2) optionally a pharmacodynamic monitoring module.
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